Mutational, Structural, and Kinetic Evidence for a Dissociative Mechanism in the GDP-Mannose Mannosyl Hydrolase Reaction
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- 作者：Zuyong Xia ; Hugo F. Azurmendi ; Luke L. Lairson ; Stephen G. Withers ; Sandra B. Gabelli ; Mario A. Bianchet ; L. Mario Amzel ; and Albert S. Mildvan
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：June 28, 2005
- 年：2005
- 卷：44
- 期：25
- 页码：8989 - 8997
- 全文大小：229K
- 年卷期：v.44,no.25(June 28, 2005)
- ISSN：1520-4995
文摘
GDP-mannose hydrolase (GDPMH) catalyzes the hydrolysis of GDP-
-D-sugars by nucleophilicsubstitution with inversion at the anomeric C1 atom of the sugar, with general base catalysis by H124.Three lines of evidence indicate a mechanism with dissociative character. First, in the 1.3 Å X-ray structureof the GDPMH-Mg2+-GDP·Tris+ complex [Gabelli, S. B., et al. (2004) Structure 12, 927-935], theGDP leaving group interacts with five catalytic components: R37, Y103, R52, R65, and the essentialMg2+. As determined by the effects of site-specific mutants on kcat, these components contribute factorsof 24-, 100-, 309-, 24-, and 
105-fold, respectively, to catalysis. Both R37 and Y103 bind the 
-phosphateof GDP and are only 5.0 Å apart. Accordingly, the R37Q/Y103F double mutant exhibits partially additiveeffects of the two single mutants on kcat, indicating cooperativity of R37 and Y103 in promoting catalysis,and antagonistic effects on Km. Second, the conserved residue, D22, is positioned to accept a hydrogenbond from the C2-OH group of the sugar undergoing substitution at C1, as was shown by modeling an-D-mannosyl group into the sugar binding site. The D22A and D22N mutations decreased kcat by factorsof 102.1 and 102.6, respectively, for the hydrolysis of GDP--D-mannose, and showed smaller effects onKm, suggesting that the D22 anion stabilizes a cationic oxocarbenium transition state. Third, the fluorinatedsubstrate, GDP-2F--D-mannose, for which a cationic oxocarbenium transition state would be destabilizedby electron withdrawal, exhibited a 16-fold decrease in kcat and a smaller, 2.5-fold increase in Km. TheD22A and D22N mutations further decreased the kcat with GDP-2F--D-mannose to values similar tothose found with GDP--D-mannose, and decreased the Km of the fluorinated substrate. The choice ofhistidine as the general base over glutamate, the preferred base in other Nudix enzymes, is not due to thegreater basicity of histidine, since the pKa of E124 in the active complex (7.7) exceeded that of H124(6.7), and the H124E mutation showed a 102.2-fold decrease in kcat and a 4.0-fold increase in Km at pH9.3. Similarly, the catalytic triad detected in the X-ray structure (H124- - -Y127- - -P120) is unnecessaryfor orienting H124, since the Y127F mutation had only 2-fold effects on kcat and Km with either H124 orE124 as the general base. Hence, a neutral histidine rather than an anionic glutamate may be necessaryto preserve electroneutrality in the active complex.
-D-sugars by nucleophilicsubstitution with inversion at the anomeric C1 atom of the sugar, with general base catalysis by H124.Three lines of evidence indicate a mechanism with dissociative character. First, in the 1.3 Å X-ray structureof the GDPMH-Mg2+-GDP·Tris+ complex [Gabelli, S. B., et al. (2004) Structure 12, 927-935], theGDP leaving group interacts with five catalytic components: R37, Y103, R52, R65, and the essentialMg2+. As determined by the effects of site-specific mutants on kcat, these components contribute factorsof 24-, 100-, 309-, 24-, and 105-fold, respectively, to catalysis. Both R37 and Y103 bind the -phosphateof GDP and are only 5.0 Å apart. Accordingly, the R37Q/Y103F double mutant exhibits partially additiveeffects of the two single mutants on kcat, indicating cooperativity of R37 and Y103 in promoting catalysis,and antagonistic effects on Km. Second, the conserved residue, D22, is positioned to accept a hydrogenbond from the C2-OH group of the sugar undergoing substitution at C1, as was shown by modeling an-D-mannosyl group into the sugar binding site. The D22A and D22N mutations decreased kcat by factorsof 102.1 and 102.6, respectively, for the hydrolysis of GDP--D-mannose, and showed smaller effects onKm, suggesting that the D22 anion stabilizes a cationic oxocarbenium transition state. Third, the fluorinatedsubstrate, GDP-2F--D-mannose, for which a cationic oxocarbenium transition state would be destabilizedby electron withdrawal, exhibited a 16-fold decrease in kcat and a smaller, 2.5-fold increase in Km. TheD22A and D22N mutations further decreased the kcat with GDP-2F--D-mannose to values similar tothose found with GDP--D-mannose, and decreased the Km of the fluorinated substrate. The choice ofhistidine as the general base over glutamate, the preferred base in other Nudix enzymes, is not due to thegreater basicity of histidine, since the pKa of E124 in the active complex (7.7) exceeded that of H124(6.7), and the H124E mutation showed a 102.2-fold decrease in kcat and a 4.0-fold increase in Km at pH9.3. Similarly, the catalytic triad detected in the X-ray structure (H124- - -Y127- - -P120) is unnecessaryfor orienting H124, since the Y127F mutation had only 2-fold effects on kcat and Km with either H124 orE124 as the general base. Hence, a neutral histidine rather than an anionic glutamate may be necessaryto preserve electroneutrality in the active complex.