Detailed Dissection of a New Mechanism for Glycoside Cleavage: α-1,4-Glucan Lyase

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Abstract
The unusual enzyme, Gracilariopsis α-1,4-glucan lyase of the sequence-related glycoside hydrolase family 31, cleaves the glycosidic bond of α-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 kcat/Km indicates two ionizable groups in the active site with apparent pKa values of 3.05 and 6.66. Brønsted relationships of log kcat versus pKa and log(kcat/Km) versus pKa for a series of aryl glucosides both show a linear monotonic dependence on leaving group pKa with low βlg values of 0.32 and 0.33, respectively. The combination of these low βlg values with large secondary deuterium kinetic isotope effects (kH/kD = 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 (Ki = 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 (kH/kD = 1.23) at C-1 and the small primary deuterium kinetic isotope effect (kH/kD = 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 α-glucosidases is clear evidence for the evolution of an enzyme mechanism within the family.

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