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Binding of substrates and inhibitors to human pancreatic alpha amylase

University of British Columbia

Human pancreatic alpha-amylase (HPA) is the enzyme responsible for hydrolyzing starch within the gut into shorter oligosaccharides. Selective inhibition targeted at only HPA could be used to modulate blood glucose levels for the treatment of diabetes and obesity. Montbretin A (MbA) is a potent (Ki = 8.1 nM) and specific inhibitor of HPA. Controlled degradation studies on MbA, coupled with inhibition analysis, identified an essential high-affinity core structure comprising the myricetin and caffeic acid moieties linked via a disaccharide, mini-MbA. X-ray structural analyses of the complex of MbA-HPA confirmed the importance of this core structure and revealed a novel mode of glycosidase inhibition wherein internal pi-stacking interactions between the myricetin and caffeic acid organize their ring hydroxyls for optimal hydrogen bonding to the catalytic residues of HPA. The simplified analogue mini-MbA therefore offers potential for new strategies for glycosidase inhibition and therapeutic development. As part of a search for selective, mechanism-based covalent inhibitors of HPA, chemo-enzymatic syntheses of oligoglycosyl epi-cyclophellitols are described. Alpha-1,4 glucosyl epi-cyclophellitol, synthesized from epi-cyclophellitol by coupling of a glucosyl moiety using maltose phosphorylase, inactivated HPA stoichiometrically. X-ray crystallographic analysis of the covalent derivative so formed confirmed its reaction at the active site with the catalytic nucleophile Asp197. Another trisaccharide analogue 4’-O-methyl-alpha-maltosyl epi-cyclophellitol was synthesized enzymatically or by in situ elongation by HPA. Both of the inhibitors showed time-dependent inactivation of HPA, with the trisaccharide version being a better inactivator. This new class of mechanism-based inhibitors will be useful as activity-based probes for amylases. Several potential starch binding sites have been identified on the surface of HPA by crystallography, but their role, if any, in starch degradation is unknown. Through analysis of the binding of HPA mutants, modified individually at each site, to soluble and granule starch, two of these surface binding sites (SBSs) were shown to play a role in starch granule binding. A quite separate site was shown to be important for binding to, and cleavage of, soluble starch. Binding at SBSs was distinguished from binding to the active site by blocking the active site with the glycosyl epi-cyclophellitol mechanism-based inactivators.

Text

2016-12-31

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/58311

Chemistry

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/