Substrate recognition by three family 13 yeast α-glucosidases -: Evaluation of deoxygenated and conformationally biased isomaltosides

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, DeltaDeltaGdouble dagger, contributed by the individual OH-groups was calculated from the activities for the parent and the deoxy analogs, respectively, according to DeltaDeltaGdouble dagger = -RT ln[(V-max/K-m)(analog)(V-max/K-m)(parent)]. This analysis of the energetics gave DeltaDeltaGdouble dagger 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 H-1 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.