Detailed Kinetic Analysis of a Family 52 Glycoside Hydrolase: A -Xylosidase from Geobacillus stearothermophilus
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- 作者：Tsafrir Bravman ; Gennady Zolotnitsky ; Valery Belakhov ; Gil Shoham ; Bernard Henrissat ; Timor Baasov ; and Yuval Shoham
- 刊名：Biochemistry
- 出版年：2003
- 出版时间：September 9, 2003
- 年：2003
- 卷：42
- 期：35
- 页码：10528 - 10536
- 全文大小：127K
- 年卷期：v.42,no.35(September 9, 2003)
- ISSN：1520-4995
文摘
Geobacillus stearothermophilus T-6 encodes for a 
-xylosidase (XynB2) from family 52 ofglycoside hydrolases that was previously shown to hydrolyze its substrate with net retention of the anomericconfiguration. XynB2 significantly prefers substrates with xylose as the glycone moiety and exhibits atypical bell-shaped pH dependence curve. Binding properties of xylobiose and xylotriose to the activesite were measured using isothermal titration calorimetry (ITC). Binding reactions were enthalpy drivenwith xylobiose binding more tightly than xylotriose to the active site. The kinetic constants of XynB2were measured for the hydrolysis of a variety of aryl -D-xylopyranoside substrates bearing differentleaving groups. The Br
nsted plot of log kcat versus the pKa value of the aglycon leaving group revealsa biphasic relationship, consistent with a double-displacement mechanism as expected for retaining glycosidehydrolases. Hydrolysis rates for substrates with poor leaving groups (pKa > 8) vary widely with theaglycon reactivity, indicating that, for these substrates, the bond cleavage is rate limiting. However, nosuch dependence is observed for more reactive substrates (pKa < 8), indicating that in this case hydrolysisof the xylosyl-enzyme intermediate is rate limiting. Secondary kinetic isotope effects suggest that theintermediate breakdown proceeds with modest oxocarbenium ion character at the transition state, andbond cleavage proceeds with even lower oxocarbenium ion character. Inhibition studies with several glucoanalogue inhibitors could be measured since XynB2 has low, yet sufficient, activity toward 4-nitrophenyl-D-glucopyranose. As expected, inhibitors mimicking the proposed transition state structure, such as1-deoxynojirimycin, bind with much higher affinity to XynB2 than ground state inhibitors.
-xylosidase (XynB2) from family 52 ofglycoside hydrolases that was previously shown to hydrolyze its substrate with net retention of the anomericconfiguration. XynB2 significantly prefers substrates with xylose as the glycone moiety and exhibits atypical bell-shaped pH dependence curve. Binding properties of xylobiose and xylotriose to the activesite were measured using isothermal titration calorimetry (ITC). Binding reactions were enthalpy drivenwith xylobiose binding more tightly than xylotriose to the active site. The kinetic constants of XynB2were measured for the hydrolysis of a variety of aryl -D-xylopyranoside substrates bearing differentleaving groups. The Brnsted plot of log kcat versus the pKa value of the aglycon leaving group revealsa biphasic relationship, consistent with a double-displacement mechanism as expected for retaining glycosidehydrolases. Hydrolysis rates for substrates with poor leaving groups (pKa > 8) vary widely with theaglycon reactivity, indicating that, for these substrates, the bond cleavage is rate limiting. However, nosuch dependence is observed for more reactive substrates (pKa < 8), indicating that in this case hydrolysisof the xylosyl-enzyme intermediate is rate limiting. Secondary kinetic isotope effects suggest that theintermediate breakdown proceeds with modest oxocarbenium ion character at the transition state, andbond cleavage proceeds with even lower oxocarbenium ion character. Inhibition studies with several glucoanalogue inhibitors could be measured since XynB2 has low, yet sufficient, activity toward 4-nitrophenyl-D-glucopyranose. As expected, inhibitors mimicking the proposed transition state structure, such as1-deoxynojirimycin, bind with much higher affinity to XynB2 than ground state inhibitors.