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Research has been conducted on the alpha-1,4-glucan lyase. The mechanistic study of this lyase which includes kinetic isotope effect measurements has been carried out and the results are discussed.

The unusual enzyme, Gracilariopsis α-l,4-glucan lyase of the sequence-related glycoside hydrolase family 31, cleaves the glycosidic bond of tx- 1,4-glucans via a β-elimination reaction involving a covalent glycosyl-enzyme intermediate (Lee, S. S., Yu, S., and Withers, S. G. (2002) J. Am. Chem. Soc. 124,4948-4949). The classical bell-shaped pH dependence of k[SUBcat]/K[SUBm] indicates two ionizable groups in the active site with apparent pK[SUBa] values of 3.05 and 6.66. Br0nsted relationships of log k[SUBcat] versus pK[SUBa] and log(k[SUBcat]/K[SUBm]) versus pK[SUBa] for a series of aryl glucosides both show a linear monotonic dependence on leaving group pK[SUBa] with low β[SUBlg] values of 0.32 and 0.33, respectively. The combination of these low β[SUBlg] values with large secondary deuterium kinetic isotope effects (k[SUBH]/k[SUBD] = 1.16 ˜ 1.19) on the first step indicate a glycosylation step with substantial glycosidic bond cleavage and proton donation to the leaving group oxygen at the transition state. Developed oxocarbenium ion character of the transition state is also suggested by the potent inhibition afforded by acarbose and 1-deoxynojirimycin (K[SUBi] = 20 and 130 nM, respectively) and by the substantial rate reduction afforded by adjacent fluorine substitution. For only one substrate, 5-fluoro-α-D-glucopyranosyl fluoride, was the second elimination step shown to be rate-limiting. The large α-secondary deuterium kinetic isotope effect (k[SUBH]/k[SUBD] = 1.23) at C-1 and the small primary deuterium kinetic isotope effect (k[SUBH]/k[SUBD] = 1.92) at C-2 confirm an E2 mechanism with strong E1 character for this second step. This considerable structural and mechanistic similarity with retaining tx-glucosidases is clear evidence for the evolution of an enzyme mechanism within the family. [ABSTRACT FROM AUTHOR]

Investigates the nucleophilic displacement of glucan lyase. Inhibitors of α-glucosidase; Formation of a covalent glucosyl-enzyme intermediate; Mechanism of hydration of glycals.