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Biochemistry. 2006 Jan 17;45(2):571-80.

Mechanistic analysis of the unusual redox-elimination sequence employed by Thermotoga maritima BglT: a 6-phospho-beta-glucosidase from glycoside hydrolase family 4.

Author information

1
Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z1.

Abstract

"Classical" glycosidases utilize either direct or double-displacement mechanisms involving oxocarbenium ion-like transition states to catalyze the hydrolysis of glycosidic bonds. By contrast, the mechanism of the glycosidases in glycoside hydrolase family 4 has been recently proposed to involve NAD+-mediated redox steps along with alpha,beta-elimination and addition steps via anionic intermediates. Support for this mechanism in BglT, a 6-phospho-beta-glucosidase in family 4, has been provided through mechanistic and X-ray crystallographic analyses [Yip, V. L.Y., et al. (2004) J. Am. Chem. Soc. 126, 8354-8355] in which primary deuterium kinetic isotope effects for the hydride abstraction at C3 and for the alpha-proton abstraction at C2 indicate that these two steps are both partially rate-limiting. Current data reveal that there is no secondary deuterium kinetic isotope effect associated with the rehybridization of the C1 sp3 center to a sp2 center. Furthermore, a flat linear free energy relationship was established with a series of aryl 6-phospho-beta-D-glucosides of varying leaving group abilities. Taken together, these data indicate that cleavage of the C1-O1 linkage does not occur during a rate-limiting step. Since the deprotonation at C2 is slow and partially rate-limiting while the departure of the leaving group is not, a stepwise E1(cb)-type mechanism rather than an E1 or a concerted E2-syn mechanism is proposed. Direct evidence for the role of NAD+ was obtained by reduction in situ using NaBH4 leading to an inactive enzyme that could be reactivated by the addition of excess NAD+. This was accompanied by the expected UV-vis spectrophotometric changes.

PMID:
16401086
DOI:
10.1021/bi052054x
[Indexed for MEDLINE]

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