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

Abstract

The unusual enzyme, Gracilariopsis alpha-1,4-glucan lyase of the sequence-related glycoside hydrolase family 31, cleaves the glycosidic bond of alpha-1,4-glucans via a beta-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(cat)/K(m) indicates two ionizable groups in the active site with apparent pK(a) values of 3.05 and 6.66. Brønsted relationships of log k(cat) versus pK(a) and log(k(cat)/K(m)) versus pK(a) for a series of aryl glucosides both show a linear monotonic dependence on leaving group pK(a) with low beta(lg) values of 0.32 and 0.33, respectively. The combination of these low beta(lg) values with large secondary deuterium kinetic isotope effects (k(H)/k(D) = 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(i) = 20 and 130 nM, respectively) and by the substantial rate reduction afforded by adjacent fluorine substitution. For only one substrate, 5-fluoro-alpha-D-glucopyranosyl fluoride, was the second elimination step shown to be rate-limiting. The large alpha-secondary deuterium kinetic isotope effect (k(H)/k(D) = 1.23) at C-1 and the small primary deuterium kinetic isotope effect (k(H)/k(D) = 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 alpha-glucosidases is clear evidence for the evolution of an enzyme mechanism within the family.

A Historical Perspective for the Catalytic Reaction Mechanism of Glycosidase; So As to Bring about Breakthrough in Confusing Situation

“…16C appears to be rational. The hydration to the oxonium ion inter- [113][114][115] Pathway (C) is newly postulated in this review.…”

“…113) This suggests that the reaction mechanism of -1,4-glucan lyase is closely correlated with that of -glucosidase. Two reaction mechanisms [113][114][115] for lyase have been proposed, as shown in Fig. 16A and B.…”

An enzyme family reunion — similarities, differences and eccentricities in actions on α-glucans

“…Dikstra & S. Yu, personal communication) insight into specificity determinants of this enzyme family will expand. The structural information complements kinetics analysis contributing to explain the structural basis for the different reaction mechanisms of the starch lyases and the hydrolases (Lee et al 2003). A bootstrap diagram (Fig.…”

Type 2 Diabetes Mellitus- Disease, Diagnosis and Treatment

“…AGIs such as acarbose, voglibose and miglitol are pseudo-carbohydrates that competitively inhibit α-glucosidase enzymes located in the brush border of small intestine that hydrolyze non-absorbable polysaccharides and oligosaccharides into absorbable monosaccharides [40]. As a result, the effect of these drugs is to retard glucose absorption after a meal, and consequently lowers postprandial insulin levels and hyperglycemia peaks in patients with few β-cell reserves.…”