Video game live streaming is a kind of real-time video social media that integrates traditional broadcasting and online gaming. With the rapid popularity of video game live streaming in the past decade, researchers have started to investigate the relationship between the use of video game live streaming and various psychological variables. In order to fully understand the factors that affected user participation (streamers and audiences) in video game live streaming and provide a reference to the mental health issues of Internet addiction, this paper summarizes the relevant literature on user behavior in video game live streaming. First, we comprehensively searched literature in six social science databases and thus obtained 24 papers that meet our inclusion criteria. Second, the above literature was presented in table form for classification and we found that the effect factors of user behavior in video game live streaming mainly include user demands and platform impact. Based on Use and Satisfaction theory, this paper reviewed the following four aspects: streamer demand, audience demand, interaction behavior and platform impact, then a relevant theoretical framework was constructed. Finally, this paper looks forward to possible future research topics based on the research platform, research data and research content and so on, hoping to provide a foundation and new ideas for future research. In rePacific) . By 2018Video game live streaming users can be divided into streamers and audiences. The main difference between them is that the former produce information or content, while the latter receives or consumes it . The strIn summary, the literature was reviewed from the following four aspects: streamer demand, audience demand, user interaction behavior, and platform impact; and the relevant theoretical framework was constructed. Finally, the paper looks ahead at the future research directions. Through this literature review, we hope to provide a clearer picture of the current research state of the factors, which impacted user participation in video game live streaming. Also, we hope to provide a reference for the psychology of addiction to video game live streaming.Literature retrieval refers to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines . The proThis paper adopts the following steps to retrieve and collect existing literature on video game live streaming. First, through literature search, we gathered various expressions of video game live streaming and determined the retrieval keywords: “live streaming,” “Internet broadcast,” “network broadcast,” “webcast,” “video game,” “online game,”. Thereafter, various combinations of these keywords were created. Next, we selected the most commonly used social science databases: Elsevier SD, EBSCO, Spring link, Emerald, Wiley Online Library, and Google Scholar. This paper takes into account that the earlier video game live streaming platform Twitch was officially founded in 2011, after which live gaming was widely used. Meanwhile most of academic researches on video game live streaming have occurred from 2012 onwards. Therefore, the search time was determined to be limited to peer-reviewed articles in English published between January 2012 and September 2019. Then, for the retrieved literature, we traced relevant studies and the referenced studies. Furthermore, a secondary search was conducted in April of 2020 to locate any additional articles published since the original search.In adherence to the PRISMA guidelines  see Fig, articleResearch content—the main matter of the literature should be about user behavior and its effect factors on video game live streaming, including when using platforms such as Twitch, YouTube Gaming, DouYu TV, Huya.com, as well as other popular online game platforms. And the article must include clear live streaming user objects, research methods, research data.Date must be from January 2012 to April 2020.Article must have been written in English.Article must either be a peer-reviewed journal or a conference article.To ensure rigidity of this literature review, studies were required to meet the following inclusion criteria:A total of 24 studies were selected. These examined three main areas: effect factors on streamer participation (six papers), effect factors on audience participation (twelvepapers), and effect factors on interactive behaviors (eight papers). Notably, two of the selected twelve papers examining the effect factors of audience participation also mentioned interactive behavior. Sorting through the existing literature revealed that Kaytoue et al.  were earThis paper divided the main issues into four types: streamer demand, audience demand, user interaction behavior, and platform influence. This classification referred to studies by Scheibe  and ZimmThe existing literature mostly defines video game live streaming based on form and content. With regard to form, video game live streaming is described as a new media that integrates traditional broadcast and online gaming ,2, such In terms of content, video game live streaming is defined as a network broadcast with “online games” as the specific content ,15, suchVideo game live streaming users are divided into two groups, streamer and the audience, and the differences between them are related to information production and reception . StreameThere are three main features of video game live streaming mentioned in the existing literature:First, live streaming is performed in real-time; that is, streamers share online videos of playing game  while auSecond, the activity is highly conducive to sociability; that is, live streaming is a type of social media ,17,19. SThe third feature is the dissemination of suspense; that is, compared to traditional media , video game live streaming has the characteristics of suspense. For example, online games and other activities with a competitive nature encapsulate high moments, low moments, and a suspenseful result, whereas when watching pre-recorded media, the result is fait accompli, making content less engaging because of spoilers or a pre-determined outcome . Wulf etIt has been mentioned in existing literature that there are three main features of video game live streaming: real time, social media and suspense compared to other video platforms and social platforms. Therefore, these three characteristics should be fully taken into account when investigating the factors that affect user behavior in video game live streaming.According to different types of games, communities of video game live streaming can be divided into three categories: Let’s Players (self-entertainment), Speedrunner (quickly complete a game), and E-sports  ,25. AmonSocial integration was a demand to strengthen ties with relatives, friends, and family . AccordiPersonal integration was a demand to improve personal confidence, credibility, and status , and maiAffection demand is an emotional, pleasurable and aesthetic experience . That isIn addition to the main factors of the aforementioned three demands, external rewards are also factors on participating in live streaming. External rewards are divided into reward measures of the platform  and rewards from the audience . Research showed that external rewards had positive effects on game players to continuously streaming ,19,27. FSocial integration mainly refers to the social demand of the audience, including the connection between audiences and streamers, the audience and audience groups (other audiences). First, social interaction (with other audiences) was an important factor that impact audiences watching live streaming . Hu et aPersonal integration mainly comprises the audience’s self-presentation and self-recognition demand. Sjoblom et al.  found thTension release is inferred a demand for escape and diversion of attention . That isThe cognitive aspect infers to satisfying a demand for acquiring knowledge, information, and understanding . That isAffective demand mainly includes suspense, excitement, entertainment, and interest. Lim et al.  thought In addition to major factors that satisfy the five aforementioned demands, other influencing factors include the audience’s preference of content. Chen and Pan  analyzedParasocial interaction, proposed by Horton and Richard Wohl , was an Live streaming is a medium combining real-time video and text chat ,41, inteAudiences are willing to invest not only time, but also money on the live streaming platform. Some audiences choose to pay a reward or subscribe to streamer by spending more money. Some scholars believe that these members can receive unique benefits from the streamer. Wulf et al.  and GrosStreamers, viewing duration and the number of audience members affect the audience’s intention to reward. Gros et al.  found thAudiences giving rewards and gifts have the characteristics of time and inclination. Karhulahti et al.  made an Unlike the traditional learning model—where only the apprentice is learning—the existing literature suggests that learning in video game live streaming is involved on both sides. Video game live streaming facilitates learning interaction between the streamer and the audience. Streamers and audiences play roles of mentors and apprentices, respectively . Wulf etStreamer’s mentorship skills and learner’s character will affect the learning of the users on video game live streaming. Based on the cognitive load theory, Payne et al.  found thThe video game live streaming platform is an important factor influencing the participation of both audiences and streamers. Features of video game live streaming platforms include website attractiveness, gamification mechanisms, socialization, convenience, digitalization and so on, all of these features attract streamers and audiences to participate in live streaming. The following section reviews the impact of live gaming platforms on participation behaviors of live broadcasters and audiences.The attractiveness of the website can impact streamers’ behavior, which includes the website’s graphic design, user interface, and live channel. These factors had a positive impact on the participation of streamers . GandolfThe design of gamification mechanisms on the live streaming platform will affect the behavior of continuous live streaming. Gamification is the use of game design elements in non-game environments, products, and services to stimulate user expectations, such as points, badges, or money . SjoblomIn summary, features of video game live streaming platform would attract streamers to participate in live streaming such as website’s graphic design, user interface, and live channel, gamification mechanism. Therefore, these platform features mentioned above should be considered when studying streamers’ addiction to video game live streaming.The design of gamification mechanisms and socialization can affect the audience’s participation in the live streaming. Subscriptions, rewards, and game mechanism design, along with the sociability of the chat room design on the platform can meet the audience’s social demands. Fietkiewicz and Stock  proposedThe digital experience and convenience on the platform can also affect the audience participation. Neus et al.  used theThe gamification mechanics and socialization of live streaming platforms meet the social demands of the audience. And the digital experience and convenience better satisfy the viewing experience of the audience. In conclusion, features of the live streaming platform would attract audiences to participate in the live streaming, including gamification mechanisms, socialization, digital experience and convenience. Therefore, these platform features mentioned above should be considered when studying the audience’s addiction to video game live streaming.Video game live streaming platforms are becoming more and more popular, with both streamers and audiences spending more time on these platforms. It has become a matter of concern which factors affect user participation in video game live streaming. This paper analyzed the existing literature in terms of both user demands and platform characteristics, and found that user demands in the five major aspects of social integration, personal integration, tension release, affection, and cognition affect user behavior in the live streaming. This provides a comprehensive analysis of these research current state about user engagement behavior in video game live streaming. Furthermore, there are related implications in terms of users indulging in video game live streaming.As with game obsession, audience and streamer obsession with live gaming may be a mental health issue of concern. By summarizing the factors that impact the user behavior, this paper found these factors that might affect addiction to video game live streaming from two aspects, namely, user demands and platform impact. For streamers, social integration, personal integration, affective and additional rewards should be fully considered. For audiences, attention should be paid to affecting factors such as social integration, personal integration, tension release, affective demand, and cognition, and game category. Of these, social and affective demands are the more important impact factors for both streamers and audiences. And for cognitive demands, no relevant factors have been found to affect the behavior of streamers. The audience wants to learn more about game skill through the platform, but there is not much desire to satisfy the cognitive demands. Therefore, this paper argues that cognitive demand is unlikely to be a factor in user addiction to video game live streaming. In addition, platform features such as website attractiveness, gamification mechanisms, socialization, convenience, digitalization, and so on, should be highlighted in the factors of user addiction to video game live streaming.Although some valuable results have been achieved in existing research, further research and improvement are expected due to the fact that user behavior in video game live streaming is relatively lacking in the field of mental health research. This paper proposes that future studies might explore the following aspects: First, expansion of the research platform. At present, more than half of the research on video game live streaming was based on the Twitch platform. Previous studies have found that existing characteristics of video game live streaming platforms would affect users’ participation behaviors. On this basis, research should be extended to other platforms—perhaps those in different countries—to explore the influence of personalized and differentiated designs of various platforms on users. In addition, most existing video game live streaming platforms were connected to other social platforms, and users could converse with each other on these platforms. For example, Douyu allows video link sharing, therefore, both audiences and streamers can share video links to WeChat, Weibo, QQ, and other social platforms. For the sake of convenience, users are also allowed to log in directly through such social media accounts. Hence, we can explore impact of this kind of cross-platform joint approach on users of video game live streaming in the future.Second, expansion effect factor on streamer addiction. Existing literature is lacking on studying factors affecting the streamer participation. This article argues that the effect of tension release and cognitive on streamer participation should be considered. Streamers could also relax via streaming playing games by themselves. Because playing the game was recognized as a way of relieving stress and the live-streaming process allowed for conversation with the audience. In addition, it is also possible that cognitive demands of streamer could be fulfilled by streaming. Although existing research did not indicate that streamer was engaged in streaming to fulfill cognitive demand, and it was mentioned in the literature that the live streamer might also learn some gaming skills through interaction with audience during process of acting as a teacher . Thus, fThird, expansion in methods for acquiring research data. Internet data mining has become a major trend in the field of Internet behavior research. In future research, network data mining can be used to collect research data. Compared to general questionnaire surveys, interviews and other such methods, network data mining can obtain primary data more objectively and accurately. For example, Nascimento et al.  created Fourth, expansion user behavior of video game live streaming with the implementation of Tandem Play . At present, studies are largely based on users’ behavior under the condition that the streamer performs for the audience member. Scully-Blaker et al.  have defBased on the existing literature, this paper comprehensively defined the video game live streaming, outlines the characteristics and the community classification of the video game live streaming. Moreover, we analyzed the effect factors on user participation and constructed the theoretical framework. But there are also some limitations in this paper. First, a limited number of papers—only 24—were collected. This might be due to the title and keyword filtering phase, which indicated that the study generally pertained to user behavior on the live streaming and online gaming. Since only English-language studies were included in the review, some key studies in other languages might be excluded. Second, we only analyzed user behavior in video game live streaming in terms of user demand and live streaming platform characteristics. However, the factors impacting users’ participation in video game live streaming are more complex and other aspects such as the type of game are not considered.This paper is a systematic literature review on the impact factors of user engagement in video game live streaming. At present, no similar literature review is found in the field of users’ addictions to the video game live streaming. According to the 24 relevant literature references, we summarized the definition, features, community classification, and users of video game live streaming. Then, we analyzed these papers separately from factors of live streamer, audience, and interactive behavior. Among these, streamer interaction behavior includes chatting and learning. Audience interaction behavior includes chat interaction, reward and subscription, and learning interaction. From the perspective of the platform and two user groups, this paper constructed a framework of effect factors of user engagement in video game live streaming. It was found that effect factors on video game live streaming user behavior mainly involve two aspects: user demands and platform influence. Video game live streaming can meet user demands in social integration, personal integration, tension release, affection, and cognition. The website attractiveness, gamification mechanisms, socialization, convenience, digitalization and other features of the platform would attract users to participate in live streaming. We hope these findings can help address the mental health issues of video games live streaming addiction. Furthermore, this article summarized future research directions from existing research limitations such as research platform, effect factors, research methods, user behavior. This article hopes to provide ideas for future research.


Gender differences in alcohol use are more substantial among early adolescents in China than in the United States, presumably because of more permissive drinking norms for boys than girls in Chinese culture. This study tested a theory that gender differences in early experimentation with alcohol can be reduced through general parenting practices. Whereas traditional research has identified mediators of gender differences in alcohol use, the current research isolated moderators of gender differences and developed their implications for prevention programs.n = 8805 middle school students in four cities). Youth completed anonymous surveys in classroom settings. The study examined interaction effects between gender and parenting variables using multiple regression with robust standard errors.The study analyzed the data from the China Global School-Based Student Health Survey (Early adolescent boys exhibited higher levels of drinking than girls for all drinking outcomes. The gender differences in drinking were negatively associated with the level of perceived parental monitoring, parental involvement in adolescent school performance, and parental empathy in a nonlinear way.Results suggested that early adolescents’ perceptions of general parenting practice nonlinearly moderated gender disparities in alcohol use. Alcohol use is a leading risk factor contributing to the global disease burden in 10- to 24-year-olds (7% of disability-adjusted life-years) , and othAlthough alcohol use prevalence rates do not differ much by gender until youth reach young adulthood in the United States and most European countries –11, suchScholars have offered explanations for the gender gap in adolescent drinking patterns and drinking progression when such differences occur, with one of the most prominent explanations being a “double standard” for drinking for boys and girls based on traditional gender roles and stereotypes , 13. GreAlthough identification of mediators of gender differences  is important, it also is crucial to understand moderators of the relationship between gender and adolescent drinking, i.e., identifying contexts and situations where both genders tend to abstain from drinking compared to contexts where boys drink more than girls. The present research focuses on the family context. Parents play key roles in socializing adolescents. Relationships between parent variables  and adolescent drinking have been well documented . ParentiIn contrast to these studies conducted on youth from Western countries, limited research has examined questions relating gendered drinking to the family context in Chinese youth. Alcohol-related research with Chinese youth has been largely epidemiological, emphasizing drinking patterns , 24 and This study investigated three modifiable parenting dimensions—parental monitoring, parental involvement in adolescent school performance, and parental empathy—as possible moderators of gender differences in alcohol use. The study focused on adolescent perceptions of these constructs rather than parental behavior per se because there is considerable research to indicate that adolescent reports of parenting behavior are far more predictive of youth problem behaviors than parent reports of parenting behavior –31. ConsAdolescents’ perceived parental monitoring is negatively associated with their drinking behaviors in general –20. In oParental empathy can maintain a sense of connection with parents, and alleviate stress, which in turn can serve as a protective factor for adolescent drinking . In ChinResearch suggests that parental involvement with their children tends to buffer against the effect of mental distress on alcohol use . For perIn sum, the current research explored gender differences in early alcohol experimentation and consumption among Chinese adolescents. It explored how family contexts can potentially enhance or diminish such gender differences, focusing on three modifiable parenting constructs, namely adolescents’ perceived parental monitoring, parental empathy, and parental involvement in school performance. The research documents gender differences in adolescent alcohol consumption as a function of family contexts and has potential implications for the design of parent-based prevention programs both for youth in China and Chinese immigrant youth overseas.This study involved secondary data analysis of the China Global School-Based Student Health Survey (GSHS) , 40. We China GSHS used a two-stage cluster sampling approach . During Questions were developed based on the Youth Risk Behavior Survey questionnaire, which has been used to assess priority health-risk behaviors among 9th- to 12th-grade students in the United States biennially since 1991 . Youth RAlcohol use was measured by four items focused on the frequency of alcohol use, the amount of alcohol use, episodes of heavy drinking, and problems associated with alcohol use. Frequency of alcohol use was measured as the “number of days you had at least one drink containing alcohol during the past 30 days” using a 7-point scale from “0 days,” “1 or 2 days,” “3 to 5 days,” “6 to 9 days,” “10 to 19 days,” “20 to 29 days,” and “All 30 days.” Amount of alcohol use was measured as the “number of drinks you usually drink per day on the days you drank alcohol during the past 30 days” using a 7-point scale from “did not drink,” “less than one drink,” “1 drink,” “2 drinks,” “3 drinks,” “4 drinks,” and “5 or more drinks.” Episodes of heavy drinking were defined as “the number of times you drank so much alcohol you got really drunk,” scored on a 4-point scale from “0 times,” “1 or 2 times,” “3 to 9 times,” “10 or more times.” Problems associated with alcohol use were measured as “the number of times you ever had a hangover, felt sick, got into trouble with your family or friends, missed school, or got into fights as a result of drinking alcohol” on the same metric with that of heavy drinking. All the values were recoded to represent the midpoints of each category .Self-reports of alcohol use, of course, are subject to bias. The fact that the data were collected under conditions of anonymity and did not require face-to-face reporting of alcohol use increases their likely validity . The valnever, 1 = rarely, 2 = sometimes, 3 = most of the time, and 4 = always). Perceived parental monitoring was measured by asking: “During the past 30 days, how often did your parents or guardians really know what you were doing with your free time?”; perceived parental empathy was measured by asking: “During the past 30 days, how often did your parents or guardians understand your problems and worries?”; and parental involvement in adolescent school performance was measured by asking: “During the past 30 days, how often did your parents or guardians check to see if your homework was done?” Although criticisms of single-item measures are common, a surprisingly large number of studies have found that multiple-item scales of a construct often predict no better than single-item representations of the same construct [The China GSHS had three questions on protective parenting, requiring students to choose from categories on a 5-point metric . As such, collinearity was not an issue and the three parenting perceptions had considerable unique variance that merited treating them separately.p < .05) higher in male than female adolescents for both the entire sample and current drinkers. For the parenting behaviors, the modal response for perceived parental monitoring was “most of the time” (25.2% response rate); for parental involvement in adolescent school performance it was “sometimes”; for parental empathy it was “rarely” (24.3%). Gender differences in perceived parental monitoring and parental empathy were negligible. Male adolescents had higher levels of perceived parental involvement than female adolescents.Table p < .05).Table A second noteworthy trend in Fig. Table The present research replicated prior studies that have found gender differences in drinking by Chinese youth but in a more comprehensive way by considering distinct drinking outcomes and using large representative samples of urban youth. We found that early adolescent Chinese boys tended to drink more frequently, consume more when they drank, have more episodes of heavy drinking, and experience more problems associated with drinking compared to early adolescent Chinese girls. The consistency of the gender differences across outcomes is striking.We evaluated the boundary conditions of the gender differences under the general hypothesis that they would be negatively associated with the three facets of positive parenting, namely adolescent construals of parental monitoring, parental involvement in adolescent school performance, and parental empathy. The logic models underlying our hypotheses were grounded in adolescent perceptions about parenting behavior rather than parenting behavior per se. Adolescent perceptions are likely influenced by actual parenting behavior, but research suggests it is adolescent construals of parenting behavior that matters most –31. The An interesting puzzle that emerged in the study was the relative lack of association between drinking dynamics and the different levels of attributions of positive parenting for adolescent girls. For the most part, the levels of perceived positive parenting were not associated with female drinking, only male drinking. Although this cannot be entirely attributed to matters of base rates, floor effects undoubtedly come into play given the relatively low base rates for girls shown in Table Another important finding of the current research is that each of the parenting dimensions moderated gender differences in its own right, independent of the other parenting variables. The intercorrelations among the three parenting variables were modest and the fact that each was implicated in drinking dynamics while holding the others constant suggests each makes a unique contribution. Most research on these dimensions has focused on them in isolation rather than in a multivariate parenting context. It was useful to learn that even when considered in multivariate analyses, each of the dimensions contributed unique explained variance in its own right.For parental monitoring, adolescents’ perceived threat of being caught in a transgression as a result of presumed parental monitoring likely outweighed the social norms that boys’ drinking is more acceptable than girls’ drinking, thereby homogenizing gender differences in drinking; boys showed lower levels of alcohol use with higher levels of parental monitoring, eventually exhibiting similar drinking patterns as girls. For parental empathy, higher levels of felt empathy and understanding likely increase the likelihood that adolescents will seek help and support from their parents when they experience stress and difficulties. To the extent that drinking is perceived as a coping strategy for stress , the intTheoretically, the present study represents a significant contribution to research traditions that seek to understand gender differences in adolescent risk behaviors more generally and alcohol use in particular. By focusing on moderators rather than mediators of gendered drinking, the research elucidated how the family context can potentially constrain the impact of gendered mediators  and helped establish boundary conditions on those mediators. The research should thus foster more comprehensive analyses of gender dynamics that encourage the incorporation of both mediators and moderators into theoretical explanations of gender differences.From a prevention perspective, the present research suggests that prevention programs addressing parental monitoring, parental empathy and support, and parental involvement with school performance may be influential for adolescent Chinese boys but less so for adolescent girls. Perhaps addressing these family variables will further reinforce low drinking rates for Chinese girls, but in terms of changing alcohol use, the potential effects of the perceived parenting variables seem limited to Chinese boys. Despite this, meta-analytic results suggest that parent-based programs aimed at increasing general and/or alcohol-specific parenting skills can prevent or reduce alcohol use among both boys and girls in Western countries including Asian American girls .Intervention research on parent-based interventions in the United States support the malleable character of parenting behaviors –58. A stThe data we analyzed were collected in 2003, which raises the possibility that gender differences in drinking may have changed since then. However, more current data suggests that the gender difference in drinking still exists in China . Our intThe current study had notable strengths as well as limitations. First, the study used large representative samples of early adolescents in four major cities in China, which increases the generalizability of its findings. Second, the study was comprehensive in that it studied four distinct drinking outcomes and found similar trends with respect to each outcome. Third, the study was innovative in exploring non-linearity in outcomes as a function of the  parenting dimensions. This method allowed us to identify important trends in the data that would have been masked by applying more traditional linear modeling. Fourth, our study focuses on adolescents’ perceived parenting behavior rather than parenting behavior per se. Parents’ self-reported behavior is less predictive than adolescents’ perceptions of parenting in relation to adolescent risk behaviors including alcohol use –31. MoreAmong the limitations, the study did not include rural youth, so its results can only be generalized to urban youth. Second, given the nature of the assessment environment, the questions surrounding attributions of parental behaviors were somewhat limited by using single-item measures. Nevertheless, the parenting measures were predictive in theoretically coherent ways, which provides reassurance for the validity of the measures. Third, the study relied on adolescents’ self-reports of their alcohol use behavior, which also suggests caution in result interpretation. Self-reports, for example, can be subject to self-presentation bias. To minimize this, the survey was anonymous, voluntary, and was accompanied by instructional sets that emphasized the importance of truthful responding. Although we doubt there was systematic bias, it can’t be ruled out definitively. Fourth, the modeling is subject to omitted variable bias and must be interpreted with caution. For example, parental alcohol use could influence both adolescent drinking and adolescent perceptions of perceived parenting behavior . Parent To conclude, Chinese male adolescents exhibited higher levels of drinking frequency, drinking amount, heavy drinking, and problems associated with drinking than female adolescents. Gender differences in these drinking outcomes were non-linearly and negatively associated with perceived parental monitoring, parental involvement in adolescent school performance, and parental empathy. The reduced gender differences were mainly due to lower levels of drinking for male adolescents being associated with more positive parenting attributions rather than for female adolescents. For the latter, the associations were negligible. Parent-based alcohol use prevention and intervention programs should take gender differences into account.


We first examined the roles of Mg2+ by kinetic analysis of single nucleotide incorporation catalyzed by HIV reverse transcriptase. We show that Mg.dNTP binding induces an enzyme conformational change at a rate that is independent of free Mg2+ concentration. Subsequently, the second Mg2+ binds to the closed state of the enzyme–DNA–Mg.dNTP complex (Kd = 3.7 mM) to facilitate catalysis. Weak binding of the catalytic Mg2+ contributes to fidelity by sampling the correctly aligned substrate without perturbing the equilibrium for nucleotide binding at physiological Mg2+ concentrations. An increase of the Mg2+ concentration from 0.25 to 10 mM increases nucleotide specificity (kcat/Km) 12-fold largely by increasing the rate of the chemistry relative to the rate of nucleotide release. Mg2+ binds very weakly (Kd ≤ 37 mM) to the open state of the enzyme. Analysis of published crystal structures showed that HIV reverse transcriptase binds only two metal ions prior to incorporation of a correct base pair. Molecular dynamics simulations support the two-metal ion mechanism and the kinetic data indicating weak binding of the catalytic Mg2+. Molecular dynamics simulations also revealed the importance of the divalent cation cloud surrounding exposed phosphates on the DNA. These results enlighten the roles of the two metal ions in the specificity of DNA polymerases.Magnesium ions play a critical role in catalysis by many enzymes and contribute to the fidelity of DNA polymerases through a two-metal ion mechanism. However, specificity is a kinetic phenomenon and the roles of Mg The coordinated action of two metal ions, water molecules, and several surrounding acidic residues helps to stabilize the transition state  belongs to the A family of moderate- to high-fidelity enzymes. It serves as a good candidate for studying the two-metal-ion mechanism because kinetic characterization of single nucleotide incorporation has established the mechanistic basis for polymerase fidelity and has defined the role of a nucleotide-induced conformational change step in specificity  divided by the Kd for the weak binding of nucleotide to the open state of the enzyme (kcat/Km= K1k2). The closed state traps the nucleotide and aligns catalytic residues to facilitate fast catalysis. Under the conditions used in this study, the release of Mg.PPi is fast, so the data collected for the forward reaction cannot define k−3, the reverse of chemistry. With an RNA template, release of Mg.PPi is slow, so the chemical reaction approaches an equilibrium to provide an estimate of k−3based on the concentration dependence of the amplitude of the reaction  simulations to view the binding of Mg2+ to multiple sites on the enzyme–DNA complex. The results from MD simulations are consistent with experimental measurements of binding affinity and provide molecular details for aspects of Mg2+ binding that cannot be observed directly.Although it is clear that Mg.dNTP is the substrate for the reaction, the roles of the second Mgopen to the closed state. The second Mg2+ binds after the conformational change, stabilizes the closed state, and stimulates the chemical reaction. Accordingly, we will refer the second metal ion as the catalytic Mg2+ to distinguish it from the nucleotide-bound Mg2+, although it must be clear that both metal ions are required for catalysis. In the course of performing these experiments, we also developed a simplified method to accurately define concentrations of free Mg2+ and Mg.dNTP in solution using a Mg-EDTA buffer.The studies performed here provide new insights toward understanding the role of metal ions in DNA polymerase fidelity. We show that Mg.dNTP is necessary and sufficient to induce the conformational change from the 2+ in catalysis and specificity, we systematically studied the effects of free Mg2+ concentration on each step of the nucleotide incorporation pathway outlined in K1), forward and reverse rates of the conformational change (k2 and k−2), chemistry (k3), and PPi release (k4). We began by measuring the rate and equilibrium constants governing nucleotide binding and enzyme conformational dynamics.To address the role of Mgdd) so that Mg.dTTP binds but does not react to mimic conditions used to solve crystal structures ethyl)amino)carbonyl) coumarin) on the fingers domain as described previously. The labeling provides a signal to measure the conformational changes between open and closed states . The fluructures . The fluructures .Figure 2kon.Under conditions of rapid equilibrium substrate binding, the reaction follows a single exponential with an observed decay rate  that is a hyperbolic function of the substrate concentration. At low substrate concentrations, the slope of the concentration dependence defines the apparent second-order rate constant for substrate binding, K1 and k2; rather, we only determined the apparent second-order rate constant for Mg.dNTP binding given by the product K1k2.Because binding data were collected only at low nucleotide concentrations, the data did not resolve A and C in the presence of 10 and 0.25 mM free Mg2+, respectively. It should be noted that the Kd for Mg2+ binding to nucleotide is 29 μM . As a further test, we performed equilibrium titrations . Global fitting of the two experiments at each Mg2+ concentration using the model shown in Kd = koff/kon = 1/(K1(1 + K2)) ≈ 1/K1K2, as well as the rate constants governing binding and dissociation as summarized in 2+ concentration has little effect on the net nucleotide binding affinity. In the studies described below, we resolved these two constants (K1 and k2) using the full reaction with a normal DNA primer. The experiments reported here correlate our fluorescence signal with published structures and serve as a control for the effects of the dideoxy-terminated primer on the binding kinetics. In the next set of experiments, we measured the kinetics of reaction with a normal primer.The kinetics of binding are shown at various concentrations of Mg.dTTP in 2+ concentration on each step of the nucleotide incorporation pathway, experiments to measure the kinetics of nucleotide binding and dissociation, enzyme conformational changes, chemistry, and PPi release were all fitted globally at each free Mg2+ concentration. 2+, respectively. Figure 4A shows the time dependence of the fluorescence change after mixing the enzyme–DNA complex with various concentrations of Mg.dTTP. In each single turnover experiment, the decrease in fluorescence is due to enzyme closing after nucleotide binding, whereas the increase in fluorescence is due to enzyme opening after chemistry.To measure the effects of free Mgdd–dTTP complex  with a nucleotide trap that consists of 2 μM unlabeled enzyme–DNA complex (B). Because the DNA primer was dideoxy terminated, premixing the EDdd complex with the incoming nucleotide allows the binding but not the chemistry. After the addition of the large excess of the unlabeled enzyme–DNA complex, the rate of the fluorescence change defines the rate for the reverse of the conformational change, allowing rapid release of the nucleotide. Because these data were fit using computer simulation and we included the known kinetics of the nucleotide binding to the enzyme–DNA nucleotide trap, it was not necessary to repeat this experiment at multiple concentrations of the nucleotide trap. These results define Mg.dNTP binding and release and agree with the measurement of the net equilibrium constant shown in B and D.To measure the rate of the reverse of the conformational change, the nucleotide dissociation rate was measured by rapidly mixing a preformed enzyme–DNA complex B. BecausC). An enzyme–DNA complex (32P-labeled primer) was rapidly mixed with varying concentrations of incoming nucleotide. The rate of product formation as a function of nucleotide concentration was used to define the maximum polymerization rate (kcat) of the reaction and the specificity constant (kcat/Km). The data in kcat and kcat/Km using Equation In the next experiment, the rate of chemistry was measured in a rapid quench-flow assay C. An enzTo measure the rate of PPi release, a coupled pyrophosphatase/phosphate sensor assay was performed as described , 23. Bec2+ concentration on each step of the pathway, similar experiments and analyses were repeated at 1 and 0.25 mM free Mg2+ as shown in The four experiments were fit simultaneously to rigorously define kinetic parameters governing nucleotide incorporation  to get t2+ concentrations (from 0.25 to 10 mM) do not significantly affect the ground-state Mg.dNTP binding (K1) or the rate of the conformational change (k2) but greatly affect the rate of the chemistry (k3) and less so the reverse of the conformational change (or enzyme reopening) (k−2). Although it appears that Mg2+ concentrations affect the rate of PPi release, this reflects the rates of chemistry—our simulation only gives a lower limit and therefore no direct measurement of the PPi release rate was possible. Our data support the conclusion that PPi release was not rate limiting at any of the Mg2+ concentrations examined.The kinetic parameters are summarized in k2) to estimate the value at 37 °C. The enzyme–DNA complex was rapidly mixed with various concentrations of dTTP as described in Figures 4A and The experiments shown in max = k2+ k−2+ k3) at each temperature. Note that k−2 and k3 are much smaller than k2 so λmax ≅ k2. The experiments were repeated at various concentrations of free Mg2+: 0.25 mM (A), 1 mM (C), and 10 mM (E). The observed maximum decay rate (λmax) observed at each temperature was then graphed on an Arrhenius plot  and the values of k2 at 37 °C at each of three different free Mg2+ concentrations were obtained by extrapolation by linear regression. The results indicate that Mg2+ concentrations (from 0.25 to 10 mM) do not affect the rate constant for the conformational change (k2) (2+ binding from solution (at a concentration greater than 0.25 mM) is not required for the conformational change step, but it is required for the chemical reaction. By combining the rate constant for the conformational change (k2) with the estimates of k−2 from the nucleotide dissociation rate, we can calculate the equilibrium constant for the conformational change step , 1 mM C, and 10nd 10 mM E. The obnge (k2)  and 3. Tstep K2, .Figure 7Kd = 275 μM at 10 mM Mg2+, which is followed by the conformational change leading to a much tighter nucleotide binding. The net Kd for the two-step binding is defined by:The substrate, Mg.dTTP, binds initially to the open state with a relatively weak affinity, Kd for Mg.dTTP binding at 10 mM Mg2+ was 0.5 μM and increased to values of 1.0 and 1.1 μM at 1 and 0.25 mM Mg2+, respectively  and various concentrations of free Mg2+ (ranging from 0.25 to 10 mM). This concentration of Mg.dTTP is much greater than its Km, so we are measuring the rate of the chemistry step in this experiment. The rates of chemistry, measured by rapid-quench and stopped-flow fluorescence methods, were observed for each reaction and plotted as a function of free Mg2+ concentrations (A). The data were fit to a hyperbola to derive an apparent dissociation constant, Kd,app = 3.7 ± 0.1 mM, for the catalytic Mg2+. Because other known Mg2+ binding events reach saturation at much lower concentrations of Mg2+, they do not affect the measured Kd for the catalytic Mg2+. For example, the dissociation constant for the formation of the Mg.dTTP complex (28.7 μM) was 130-fold lower than the net Kd for binding of the catalytic Mg2+ (3.7 mM). Therefore, the observed concentration dependence of catalysis reflects only the binding of the catalytic Mg2+ to the closed ED–Mg.dTTP complex.The apparent binding affinity for the catalytic Mgtrations A. The daB we show the Mg2+ concentration dependence of the nucleotide dissociation rate, which we believe is limited by the rate of enzyme opening (k−2). We plotted the observed rate of nucleotide dissociation as a function of free Mg2+ concentration and fit the data to a hyperbola to provide an estimated Kd = 3.7 ± 0.2 mM. This defines the apparent Kd for the binding of Mg2+ to decrease the rate of nucleotide dissociation. The Mg2+ dependence of the nucleotide dissociation rate parallels the concentration dependence of the observed rate of the chemical reaction (A) suggesting that the two effects are due to the same Mg2+ binding event. The binding of the catalytic Mg2+ stabilizes the closed enzyme state as active site residues are aligned to carry out catalysis. Although the effect of Mg2+ binding on the rate of chemistry is profound (from <0.1 to 25 s−1), there is only a modest decrease (∼2-fold) in the rate of nucleotide dissociation as the Mg2+ concentration is increased from 0.25 to 10 mM.In Figure 8reaction A suggestk2) is independent of free Mg2+ concentration. To further test this postulate, a preformed EDdd complex (100 nM MDCC-labeled HIV-RT and 150 nM 25ddA/45 nt DNA) was rapidly mixed with either 50 μM dTTP or Mg.dTTP. The change of fluorescence upon dTTP or Mg.dTTP binding was monitored by stopped flow methods. In each experiment, we added 50 μM dTTP, but the free Mg2+ concentration was controlled using EDTA to allow the formation of 50 μM Mg.dTTP or ∼0 μM Mg2+ to give free dTTP. The results showed that Mg.dTTP but not dTTP induces the conformational change of HIV-RT (A–B). However, one could still argue that the trace of Mg2+ needed to form Mg.dTTP could influence the observed conformational change kinetics. As a further test, we examined the kinetics of the fluorescence change after adding Rh-dTTP, an exchange-inert metal-nucleotide complex. In the absence of free Mg2+, 50 μM Rh-dTTP induced a decrease of the fluorescence (C), although slightly lower in rate and amplitude when compared with Mg.dTTP. These results indicate that the metal–nucleotide complex is sufficient to induce enzyme closing. However, the lower rate and amplitude seen with Rh-dTTP compared with Mg.dTTP reveals differences between the two metal ion complexes. These results support conclusions derived from analysis in 2+ does not alter the rate of the conformational change step.Our results imply that the Mg.dTTP alone is sufficient for nucleotide binding and enzyme closing because the rate of conformational change . The experiment was performed by first mixing the ED complex (100 nM MDCC-labeled HIV-RT and 150 nM 25/45 nt DNA) with 10 μM Mg.dTTP in the presence of 25 μM free Mg2+ for 0.2 s (t1), followed by a second mixing with a large excess of free 10 mM Mg2+ (t2). The fluorescence change upon the second mixing was monitored by stopped-flow fluorescence to measure the opening of the enzyme after chemistry. During the first mixing step at 25 μM free Mg2+ the half-life of dTTP incorporation was >4 s (D); therefore, chemistry did not occur significantly during the first mixing step of 0.2 s. The apparent Kd = 3.7 mM predicts that only 0.7% of the catalytic Mg2+-binding sites will be occupied at 25 μM Mg2+. During the second mixing with the addition of a large excess of free Mg2+ (10 mM), the catalytic Mg2+ binds to HIV-RT and stimulates catalysis, which is followed by rapid opening of the enzyme to give a fluorescence signal. These data demonstrate that Mg.dTTP is sufficient to induce enzyme closing without the catalytic Mg2+. If the catalytic Mg2+ were required for the closing of the enzyme, we would have observed a decrease in fluorescence followed by an increase after adding excess Mg2+ in the second mixing step. The immediate reopening of the enzyme directly after the second mixing (D) demonstrates that Mg.dTTP alone is sufficient to induce the conformational change from the open to the closed state of HIV-RT. The binding of the catalytic Mg2+ in the second mixing step is necessary for fast catalysis and enzyme opening but is not required for the conformational change step. The catalytic Mg2+ binds only after enzyme closing to stimulate catalysis.Because the catalytic Mgperiment D. The exwas >4 s D; therefd mixing D demonst2+ could bind tightly to the open state of the enzyme in the absence of nucleotide, but this is never seen in crystal structures. We reasoned that, if Mg2+ binds to the open state of the enzyme–DNA complex, then it could be a competitive inhibitor of Mg.dNTP binding. A slight effect can be seen in the data in Kd for Mg.dNTP in the ground-state binding to the open form of the enzyme (1/K1) increase as the concentration of Mg2+ increases. Although we have only three data points because of the extensive analysis required to derive this number, the data can still provide an estimate of the Kd for Mg2+ binding based on the observed competition according to the following relationship:KN and KMg are the Kd values for Mg.dTTP and Mg2+, respectively, and KN,app is the apparent Kd for nucleotide binding  defines the fidelity for nucleotide incorporation in comparing a cognate base pair with a mismatch. The specificity constant is best understood as the second-order rate constant for substrate binding times the probability that, once bound, the substrate goes forward to form and release product. Steady-state kinetic parameters were calculated from the primary rate constants  is much faster than chemistry (k3), the specificity constant depends on the kinetic partitioning governed by the relative values of k−2versus k3 . If k−2 << k3, the nucleotide binding fails to reach equilibrium during turnover and the rate of chemistry does not contribute to the specificity constant; rather, it is defined only the binding and conformational change steps (kcat/Km = K1k2). As the Mg2+ concentration decreases, the rate of dissociation increases slightly, while the rate of chemistry decreases significantly. With the correct nucleotide (dTTP) incorporation at 0.25 mM Mg2+, the value of k−2 (9.7 ± 0.2 s−1) is much greater than that of k3 (0.6 ± 0.01 s−1), suggesting that the kcat/Km value is governed by the product of binding equilibrium constants and the rate of chemistry (kcat/Km = K1K2k3). When the free Mg2+ concentration was increased to 10 mM, the value of k−2 (3.9 ± 0.1 s−1) is less than that of k3 (21 ± 0.1 s−1) and therefore kcat/Km is largely governed only by the nucleotide binding (kcat/Km = K1k2). Thus, the mechanistic basis for nucleotide specificity changes as a function of the free Mg2+ concentration. We have not performed detailed analysis of the kinetics of incorporation at the lower Mg2+ concentrations because when the Mg2+ concentration is less than 0.25 mM the dNTP is not saturated with Mg2+ leading to more complex effects, including inhibition by free dNTP.The specificity constant  as shown in kcat/Km value is determined by the energy barrier between its highest peak relative to its unbound state. At 0.25 mM free Mg2+, the highest peak is the state between FDnN and FDn+1PPi (or chemistry step). Therefore, the nucleotide specificity is determined by all of the steps from its unbound state to the chemistry (kcat/Km = K1K2k3). At 10 mM free Mg2+, the highest peak is the state between EDnN and FDnN . Thus, nucleotide specificity is determined by only two steps including ground-state binding and the conformational change (kcat/Km = K1k2). At 1 mM free Mg2+, the highest peak is not obvious by inspection, and therefore, a simplified equation for defining nucleotide specificity cannot be applied. To accurately define the nucleotide specificity constant (kcat/Km), the complete equation . Mg2+ ions at the polymerase domain of HIV-RT are coordinated through polar interactions with the side chains of aspartates 110 and 185 and the three phosphates of the nucleotide substrate (B–C). In the nucleotide-bound Mg2+ (MgB in B–C), the carboxylate side chains of Asp110 and 185 as well as the two nonbridging oxygens from the phosphates of the nucleotide appear to form the four coordination interactions on the plane with a distance around 2.2 to 2.4 Å. Another phosphate oxygen and the carbonyl oxygen of valine 111 are at the apex from the opposite sides with a distance close to 2.6 Å. Therefore, MgB displays a classic octahedral coordination geometry (B). On the other hand, the catalytic Mg2+ (MgA in B–C) deviates from the standard octahedral coordination with what appears as four coordination by the side chains of Asp110 and Asp185 but not forming a plane. The two apex coordination sites are occupied by nucleotide on one end but empty on the other (C), which is presumably occupied by a solvent water molecule that is not seen in the structure. The analysis of the metal coordination indicates that the two magnesium ions are bound differently, at least as observed by the refined crystal structures.In light of our kinetic results, we analyze the published structures with the focus on the coordination and interaction around Mgatisfied A. Mg2+ iubstrate , B–C. In+ MgB in , B–C, thgeometry B. On the+ MgA in , B–C devhe other C, which 2+ ions are present indicate that the mobility of the ions varies in HIV-RT. For the Protein Data Bank ID 4PQU in which crystals were formed at high Mg2+ concentration (10 mM), MgA and MgB exhibit comparable B-factors . In other crystallization conditions in which HIV-RT was examined at lower concentrations of Mg2+ or with low-resolution diffraction, only one Mg2+ can be modeled in the density, which is consistently MgB  is defined as a measure of deviation of an atom from the average position. A high B-factor correlates to high movement and low occupancy. Within the same molecule, the B-factor can be used to estimate the relative mobility of the atoms. The B-factors when both Mgntly MgB . In partA), our solution study lacks atomic details. Structural studies provide valuable information at the atomic level but do not provide kinetic and thermodynamic parameters that define the role of Mg2+. In addition, it is naive to think that Mg2+ only binds to the tight-binding, static sites seen in the crystal structures as there are many electronegative sites available to attract positively charged ions. To fill this void and complement our kinetic studies, we performed MD simulations to gain further insights into metal ion coordination along the pathway of the DNA polymerase. Both matched and mismatched nucleotides can be studied to understand the role of metal ions in the enzyme’s function. Computer simulations of Mg2+ coordination provide molecular-level details that we cannot observe directly. However, we checked the validity of the MD simulations by comparison with what we can measure, Mg2+ binding affinities in the open and closed states, before and after Mg.dNTP binding, respectively.Despite the significant evidence from kinetic analysis and structural data listed above suggesting a weaker binding and more rapid exchange of the catalytic Mg . Details of how the ion densities were computed can be found in the Experimental Procedures section. Here, the yellow regions show local Mg2+ concentrations in the range of 2 to 10 M, whereas the regions colored in red represent concentrations greater than or equal to 40 M. Simulations suggest that a cloud of Mg2+ counter-ions surrounds the exposed DNA, as described by Manning theory ; it is highest at the center of the DNA where the DNA is most exposed to the solvent. Regions of lower concentration at 35 to 45 Å and 55 to 65 Å coincide well with the thumb-site and RNase H domains, respectively. The positively charged residues at these domains create a depletion zone for free Mg2+ ions. The important conclusion from these observations is that a significant density of Mg2+ counter-ions surrounds the DNA. These cations likely impact the binding of DNA as well as the translocation of DNA during processive polymerization. The counterion atmosphere is not seen in the crystal structures because they bind diffusively, but they are important, nonetheless.In Figure 13DNA axis B; it is 2+ ions in agreement with crystal structures as discussed in the previous section. The comparison of metal ion–binding sites with crystal structures allows us to benchmark simulation results and to further extrapolate the metal ion coordination to functional states where crystal structures are not readily available. 2+ ion coordination along the polymerase reaction pathway for matching and mismatching dNTP bound states.In addition to the diffusively bound counterions, MD simulations identify the specifically bound MgA) with local concentrations of 2+ binding to the open state is consistent with available crystal structures reporting the lack of observable Mg2+ ions in the vicinity of D110-D185 in the absence of nucleotide (2+ binding occupancy between the two aspartate residues (D110 and D185), providing a clear evidence for a dynamically changing electrostatic environment during conformational change following Mg.dNTP binding. The equilibrium positions of the nucleotide and Mg2+ ion coincide well with crystal structures  interaction with the carboxylate oxygens is a complicated topic that will be addressed in a subsequent paper.In the open state and in the absence of Mg.dNTP, two binding sites in the vicinity of D110 and D185 were observed A with locleotide . After Mructures . During  of Mg2+ , 30, 31.ructures , but the2+ coordination in a mismatched ternary complex in the closed state. The Mg2+-binding sites in a mismatched complex (Mg.dATP with template dA) are shown in Figure 14C for comparison. Unlike the matched nucleotide, the equilibrium position of the mismatch leads to misalignment of the incoming base, consistent with our previous observations , we estimated a Kd ≅ 100 mM for the site of the third metal ion. Hence, the rapid exchange of the third metal ion would make its direct observation challenging by crystallography and it is unlikely to be important under physiological conditions ([Mg2+] < 1 mM). Also, the weak binding affinity suggests that this site can be occupied by monovalent ions such as K+ or Na+ as they are more abundant under physiological conditions.Next, we studied the Mgrvations . Interes enzymes , 15. Our2+ ion coordination after the chemistry step, where the phosphodiester bond has just been formed and the by-product PPi is still bound to the complex. Similar to the previous analysis we compare the matched with a mismatched base pair (D–E). The matched nucleotide and PPi created two Mg2+-binding sites. The binding positions are similar to the ones observed before the chemistry (B). In contrast, a mismatched base accumulates three Mg2+ ions in the product complex (E), in parallel to the mismatch before the chemistry (C). Note that all simulations discussed in this section are independent runs started from random Mg2+ ion positions. One implication of our finding is that the third metal ion provides extra electrostatic stabilization to the negatively charged PPi at the active site for the mismatch. This is consistent with our observation that PPi release is slower after the incorporation of a mismatched nucleotide  provides an estimate of 3.2 kcal/mol. This agreement provides an important check for the reliability of our methodology. This also explains, for instance, that EDTA can instantly stop the polymerization reaction in our rapid-quench experiments  magnesium at the site results in misalignment of Mg.dNTP owing to the electrostatic repulsion of the two Mg2+ ions. These results suggest that the weak binding of Mg2+ (hydrated) can easily be displaced by competition with the incoming Mg.dNTP. Therefore, at physiological Mg2+ concentrations, Mg.dNTP is able to bind to the open state of the enzyme without interference by MgA. Analysis of whether MgA is hydrated or chelated by direct interaction with the carboxylate ligands is a complex problem that must be addressed by more extensive calculations.Although there is no crystallographic evidence for the tight binding of Mg Mg.dNTP . In the Kd = 28 μM), but the metal ions exchange rapidly with a half-life of 0.1 ms  and provide Mg2+ sufficient to populate the counter-ion cloud around the exposed DNA. We show kinetically that the Mg.dNTP complex binds to induce the conformational change from the open to the closed state and that the catalytic Mg2+ binds after the conformational change. Because the Mg–nucleotide complex is the natural substrate for many enzymes, the studies performed here also provide a more physiologically relevant assessment of the role of free Mg2+ ions in vivo, which may be applicable to a large number of enzymes.It has been more than 2 decades since the general two-metal-ion mechanism for phosphoryl-transfer reactions was proposed . Althougf 0.1 ms , which ck−2) versus the forward reaction (k3) is a critical factor defining nucleotide specificity (kcat/Km). Our results show that nucleotide specificity varies as a function of the free Mg2+ concentration owing to weak binding of the catalytic Mg2+ . High specificity in DNA polymerases is achieved by an induced-fit mechanism in which the enzyme closes rapidly in recognition of a correctly aligned substrate that is bound tightly as the enzyme aligns catalytic residues to facilitate the subsequent binding of the catalytic Mg2+ to simulate the chemical reaction. A mismatched nucleotide fails to stabilize the closed state or to align catalytic residues to promote catalysis, so the mismatch is released rather than react , whereas the catalytic Mg2+ binds to HIV-RT relatively weakly . The binding of Mg2+ to nucleotide (28 μM) is also much tighter (130-fold) than the binding of Mg2+ to the active site of the closed E.DNA.MgdNTP complex. Our conclusion that two Mg2+ ions bind with different affinities and in sequential order to the active site of HIV-RT during the catalytic cycle is supported by available structural data. Our molecular simulations also confirm the existence of these two binding sites. Their relative binding affinities computed from simulations are consistent with our kinetic data both in the open and closed states. High-resolution x-ray crystal structures of HIV-RT have been obtained at various stages of the reaction with enzyme in complex with nucleotide analog inhibitors  showing no obvious effect of free Mg2+ on the rate of forward conformational change (k2). In addition, the global fitting of four experiments also showed that the free Mg2+ concentration has a minimal effect on the ground-state binding (K1). These results demonstrate that catalytic Mg2+ binds after the enzyme closes. To further test this hypothesis, double mixing experiment was also performed in which Mg.dTTP was first mixed with the enzyme–DNA complex in the presence of very low free Mg2+ to allow the enzyme closing (D). After a large excess of free Mg2+ was added at the second mixing, the chemistry occurred immediately. These results further demonstrate that the catalytic Mg2+ is not required for the nucleotide-induced forward conformational change but is required for catalysis.The role of the catalytic Mg closing D. After 2+  and rate limiting  with a mismatched primer/template complex over the time scale of 4 h, suggesting an apparent rate constant of less than 10−6 s−1. These data suggest that the binding of Mg2+ and PPi does not provide sufficient energy to overcome the misalignment of the mismatched primer terminus to reach a catalytically competent state.Recently, it has been reported that the third Mg release . The ratlimiting . Furtherkcat/Km) at different Mg2+ concentrations. Our results show that the rate of nucleotide incorporation is Mg2+ dependent. The kcat/Km value for dTTP incorporation decreased approximately 12-fold as the free Mg2+ concentration was decreased from 10 to 0.25 mM , and the effects of their binding on each individual step leading to nucleotide incorporation. The studies we have performed here provided insight and detailed information about the general two-metal-ions mechanism that may be applicable to many enzymes.In conclusion, we have investigated the role of each MgN- carbamoyl]coumarin) from Sigma-Aldrich. Unreacted MDCC was removed by ion exchange using a Bio-Rex70 column. After purification, the “Coomassie Plus” protein assay was used to estimate the purified protein concentration (ThermoFisher). In addition, an active site titration was performed to determine the active site concentrations of the purified protein  using T4 polynucleotide kinase (NEB).The oligonucleotides were annealed by heating at 95 °C for 5 min, followed by slow cooling to room temperature. For making the radiolabeled primer, the 25-nt oligonucleotide was labeled at the 5’ end by γ-Rapid chemical-quench-flow experiments were performed by mixing a preformed enzyme–DNA complex (using radiolabeled DNA primer) with various concentrations of incoming nucleotide using a KinTek RQF-3 instrument . The reaction was quenched by the addition of 0.5 M EDTA at varying time points. The products were collected and separated on 15% denaturing PAGE . The results were then analyzed using ImageQuant 6.0 software (Molecular Dynamics).The stopped-flow measurements were performed by rapidly mixing an enzyme–DNA complex (using MDCC-labeled HIV-RT) with various concentrations of incoming nucleotide. The time dependence of fluorescence change upon nucleotide binding and incorporation was monitored using an AutoSF-120 stopped-flow instrument (KinTek Corp) by exciting the fluorophore at 425 nm and monitoring the fluorescence change at 475 nm using a band-pass filter with a 25-nm bandwidth (Semrock).ddDNA complex (100 nM MDCC-labeled HIV-1 wildtype RT and 150 nM DNA with a dideoxy-terminated primer) with increasing concentrations of the incoming nucleotide. The signal change was monitored continuously using the TMX titration module accessor on the SF-300x stopped-flow instrument (KinTek Corp). Fluorescence was excited at 425 nm and monitored at 475 nm using a band-pass filter with a 25-nm bandwidth (Semrock). The fluorescence signal was corrected for the small dilution during the titration.Equilibrium titration experiments were performed by titrating a 0.25 μl solution containing a preformed enzyme–KinTek Explorer software (KinTek Corp). Fitspace confidence contour analysis was also performed to estimate standard errors  using the model shown in d errors , 48.2+ concentration in solution, EDTA (500 μM) was used as a buffering system. The main equilibria that affect free Mg2+ concentration are the equilibrium constants for Mg2+ binding to dNTP, Mg2+ binding to EDTA, and the equilibrium for protonation of dNTP  u, 9 u8, 9k4 >> k3). The initial ground-state binding was modeled as a rapid equilibrium with k1 = 100 μM−1 s−1. Estimates of the remaining rate constants were then used to calculate the steady-state kinetic parameters.Steady-state kinetic parameters were calculated from the intrinsic rate constants using the following equations according to −2 opposite to a templating base adenine forming dTTP:dA pair; 3) closed-state enzyme with a mismatching nucleotide, [Mg.ATP]−2 opposite to templating DNA forming dATP:dA pair; 4) closed-state enzyme after the chemistry step where a matching nucleotide is added to the DNA strand and the pyrophosphate (PPi) group is bound; 5) closed-state enzyme after chemistry step where a mismatching nucleotide added and PPi is bound.Initial states of the ED-Mg.dNTP ternary complex were based on the open (1j5o) and closed (1rtd) state structures of HIV1-RT from the protein databank . Five in3. To neutralize the simulation box and to mimic experimental conditions we added 51 Mg2+ and 63 Cl− by randomly replacing water molecules with ions, resulting in 30 2+ in the bulk after equilibration. Water was represented by the SPC/E model  simulation for solvent equilibration. In this stage, the stiffness constant of the position restraints was decreased to 50 J/nm2 to allow local adjustments in the enzyme–substrate complex. The last frame of the trajectory was used as a starting point for unconstrained MD simulations where we compute our observables. For sampling equilibrium configurations from NVT ensemble we employed a minimum of 300-ns-long simulation in each setup described above. We removed the translational and rotational degrees of the enzyme for every 10 ps and coordinates of atom positions were recorded for each picosecond for data analysis.Following the energy minimization for each solvated system we conducted a two-step equilibration process: the first equilibration involves finding the volume of the box that gives rise to 1 atm pressure at 310K; the second equilibration allowed water and ions to equilibrate around the complex. In detail, we sampled the conformations for 2 ns from Isothermal Isobaric ensemble (NPT) using the Parrinello–Rahman scheme  and the 2+ distribution around the complex, we divided the simulation box into cubic grids of 1 Å in each direction and computed the average Mg2+ ion occupancy at each grid. From the occupancy, local ion concentrations and free Mg2+ concentration at the bulk were computed. To estimate bulk concentration, we averaged the concentrations of all grids that are 12 Å or more away from the enzyme surface.To study Mg2+ coordination to catalytic site we developed a method employing the milestoning approach. In the milestoning approach we partitioned the phase space into milestones. Using trajectory fragments, we estimated the stationary flux between milestones. Details of the milestoning approach employed can be found in Ref  and a reflecting boundary condition to R = Rbulk (reactant). Here, Rbulk is defined as the average distance between two Mg2+ if they were uniformly distributed in solution. Rbulk is ∼16 Å in 30 mM free Mg2+ concentration. As it is known that the dNTP-bound Mg is already at the active site, this distance is the average distance where one can find another Mg2+ ion. We used the association pathway that is accessible to brute force MD to compute the kinetics of the dissociation process. For dissociation rate, the milestoning equations are solved with the boundary conditions reversed.Milestoning in our study requires a reasonable pathway and a reaction coordinate that quantify the progress of the ion migration process. To obtain MgKinTek Explorer mechanism files used to fit data and refined HIV-RT structure files used for MD simulations are available upon request.K. A. J. is the President of KinTek Corp, which provided the AutoSF-120 stopped-flow, RQF-3 rapid-quench-flow, and KinTek Explorer software used in this study.