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Detailed kinetic analysis of a family 52 glycoside hydrolase: a beta-xylosidase from Geobacillus stearothermophilus.

Abstract：

:Geobacillus stearothermophilus T-6 encodes for a beta-xylosidase (XynB2) from family 52 of glycoside hydrolases that was previously shown to hydrolyze its substrate with net retention of the anomeric configuration. XynB2 significantly prefers substrates with xylose as the glycone moiety and exhibits a typical bell-shaped pH dependence curve. Binding properties of xylobiose and xylotriose to the active site were measured using isothermal titration calorimetry (ITC). Binding reactions were enthalpy driven with xylobiose binding more tightly than xylotriose to the active site. The kinetic constants of XynB2 were measured for the hydrolysis of a variety of aryl beta-D-xylopyranoside substrates bearing different leaving groups. The Brønsted plot of log k(cat) versus the pK(a) value of the aglycon leaving group reveals a biphasic relationship, consistent with a double-displacement mechanism as expected for retaining glycoside hydrolases. Hydrolysis rates for substrates with poor leaving groups (pK(a) > 8) vary widely with the aglycon reactivity, indicating that, for these substrates, the bond cleavage is rate limiting. However, no such dependence is observed for more reactive substrates (pK(a) < 8), indicating that in this case hydrolysis of the xylosyl-enzyme intermediate is rate limiting. Secondary kinetic isotope effects suggest that the intermediate breakdown proceeds with modest oxocarbenium ion character at the transition state, and bond cleavage proceeds with even lower oxocarbenium ion character. Inhibition studies with several gluco analogue inhibitors could be measured since XynB2 has low, yet sufficient, activity toward 4-nitrophenyl beta-D-glucopyranose. As expected, inhibitors mimicking the proposed transition state structure, such as 1-deoxynojirimycin, bind with much higher affinity to XynB2 than ground state inhibitors.

journal_name

Biochemistry

journal_title

Biochemistry

authors

Bravman T,Zolotnitsky G,Belakhov V,Shoham G,Henrissat B,Baasov T,Shoham Y

doi

10.1021/bi034505o

subject

Has Abstract

pub_date

2003-09-09 00:00:00

pages

10528-36

issue

eissn

0006-2960

issn

1520-4995

journal_volume

pub_type

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Detailed kinetic analysis of a family 52 glycoside hydrolase: a beta-xylosidase from Geobacillus stearothermophilus.