Important hydrogen bonding interactions between substrate OH-groups in yeast alpha-glucosidases and oligo-1,6-glucosidase from glycoside hydrolase family 13 have been identified by measuring the rates of hydrolysis of methyl alpha-isomaltoside and its seven monodeoxygenated analogs. The transition-state stabilization energy, DeltaDeltaG, contributed by the individual OH-groups was calculated from the activities for the parent and the deoxy analogs, respectively, according to DeltaDeltaG = -RT ln[(Vmax/Km)analog/(Vmax/Km)parent]. This analysis of the energetics gave DeltaDeltaG values for all three enzymes ranging from 16.1 to 24.0 kJ.mol-1 for OH-2', -3', -4', and -6', i.e. the OH-groups of the nonreducing sugar ring. These OH-groups interact with enzyme via charged hydrogen bonds. In contrast, OH-2 and -3 of the reducing sugar contribute to transition-state stabilization, by 5.8 and 4.1 kJ.mol-1, respectively, suggesting that these groups participate in neutral hydrogen bonds. The OH-4 group is found to be unimportant in this respect and very little or no contribution is indicated for all OH-groups of the reducing-end ring of the two alpha-glucosidases, probably reflecting their exposure to bulk solvent. The stereochemical course of hydrolysis by these three members of the retaining family 13 was confirmed by directly monitoring isomaltose hydrolysis using 1H NMR spectroscopy. Kinetic analysis of the hydrolysis of methyl 6-S-ethyl-alpha-isomaltoside and its 6-R-diastereoisomer indicates that alpha-glucosidase has 200-fold higher specificity for the S-isomer. Substrate molecular recognition by these alpha-glucosidases are compared to earlier findings for the inverting, exo-acting glucoamylase from Aspergillus niger and a retaining alpha-glucosidase of glycoside hydrolase family 31, respectively.
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|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
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