The hydrolysis of pullulan to panose is catalyzed by three amino acid residues within neopullulanase's active site that cleave a glycosidic bond: one glutamate and two aspartates.
 A glycosidic oxygen is first protonated by the carboxyl group of a glutamate residue (TAA Glu-230) through generic acid catalysis.
 The C1 carbon of pullulan is then attacked by a nucleophilic aspartate residue (TAA Asp-206).
 The carboxylate group of a second aspartate residue (TAA Asp-297) deprotonates an adjacent water molecule to form a hydroxide ion which hydroxylates at the C1 carbon.
 Alternatively it is possible that this reaction is concerted with the departed glycosidic oxygen being protonated to cause the hydroxylation.

The three residues responsible for neopullulanase catalysis are invariably present in enzymes of the alpha-amylase family.
 Mutation of these residues in neopullulanase results in a complete loss of enzymatic activity.

While most alpha-amylase enzymes only cleave alpha-1,4-linkages in their substrates, neopullulanase additionally cleaves alpha-1,6-linkages.
 In addition to the narrowness of the actives site resulting from the enzyme's dimeric structure, this additional functionality is thought to be facilitated by two histidine residues (TAA His-122 and TAA His-296) that interact with the glycan bond to be cleaved.
 As these histidines are present in the other alpha-amylase enzymes it is thought the functional difference arises from a difference in transition state stabilization energy contributions from the side chains of adjacent residues which vary from enzyme to enzyme.

This allows for neopullulanase's multistep breakdown of pullulan.
The enzyme first selectively hydrolyzes alpha-1,4-glucosidic bonds on the nonreducing side of pullulan's  alpha-1,6-glucosidic bonds, producing panose and panose-containing intermediates.
 These intermediates then have their alpha-1,4- and alpha-1,6-glucosidic bonds hydrolyzed to form additional panose along with smaller quantities of maltose and glucose.
The hydrolysis of pullulan to panose is catalyzed by three amino acid residues within neopullulanase's active site that cleave a glycosidic bond: one glutamate and two aspartates.  A glycosidic oxygen is first protonated by the carboxyl group of a glutamate residue (TAA Glu-230) through generic acid catalysis.  The C1 carbon of pullulan is then attacked by a nucleophilic aspartate residue (TAA Asp-206).  The carboxylate group of a second aspartate residue (TAA Asp-297) deprotonates an adjacent water molecule to form a hydroxide ion which hydroxylates at the C1 carbon.  Alternatively it is possible that this reaction is concerted with the departed glycosidic oxygen being protonated to cause the hydroxylation.
The three residues responsible for neopullulanase catalysis are invariably present in enzymes of the alpha-amylase family.  Mutation of these residues in neopullulanase results in a complete loss of enzymatic activity.
While most alpha-amylase enzymes only cleave alpha-1,4-linkages in their substrates, neopullulanase additionally cleaves alpha-1,6-linkages.  In addition to the narrowness of the actives site resulting from the enzyme's dimeric structure, this additional functionality is thought to be facilitated by two histidine residues (TAA His-122 and TAA His-296) that interact with the glycan bond to be cleaved.  As these histidines are present in the other alpha-amylase enzymes it is thought the functional difference arises from a difference in transition state stabilization energy contributions from the side chains of adjacent residues which vary from enzyme to enzyme.
This allows for neopullulanase's multistep breakdown of pullulan. The enzyme first selectively hydrolyzes alpha-1,4-glucosidic bonds on the nonreducing side of pullulan's  alpha-1,6-glucosidic bonds, producing panose and panose-containing intermediates.  These intermediates then have their alpha-1,4- and alpha-1,6-glucosidic bonds hydrolyzed to form additional panose along with smaller quantities of maltose and glucose.
Neopullulanase