|Title||Hydrogen bonding in the mechanism of GDP-mannose mannosyl hydrolase|
|Publication Type||Journal Article|
|Year of Publication||2006|
|Authors||Mildvan, AS, Xia, Z, Azurmendi, HF, Legler, PM, Balfour, MR, Lairson, LL, Withers, SG, Gabelli, SB, Bianchet, MA, Amzel, LM|
|Journal||JOURNAL OF MOLECULAR STRUCTURE|
|Date Published||JUN 5|
GDP-mannose mannosyl hydrolase (GDPMH) from E. coli catalyzes the hydrolysis of GDP-alpha-D-sugars to GDP and beta-D-sugars by nucleophilic substitution with inversion at the anomeric C1 of the sugar, with general base catalysis by His-124. The 1.3 angstrom X-ray structure of the GDPMH-Mg2+-GDP complex was used to model the complete substrate, GDP-mannose into the active site. The substrate is linked to the enzyme by 12 hydrogen bonds, as well as by the essential Mg2+. In addition, His-124 was found to participate in a hydrogen bonded triad: His-124-N delta H-... Tyr127-OH... Pro-120(C=O). The contributions of these hydrogen bonds to substrate binding and to catalysis were investigated by site-directed mutagenesis. The hydrogen bonded triad detected in the X-ray structure was found to contribute little to catalysis since the Y127F mutation of the central residue shows only 2-fold decreases in both k(cat) and K-m. The GDP leaving group is activated by the essential Mg2+ which contributes at least 105-fold to k(cat) and by nine hydrogen bonds, including those from Tyr-103, Arg-37, Arg-52, and Arg-65 (via an intervening water), each of which contribute factors to k(cat) ranging from 24- to 309-fold. Both Arg-37 and Tyr-103 bind the beta-phosphate of the leaving GDP and are only 5.0 angstrom apart. Accordingly, the R37Q/YI03F double mutant shows partially additive effects of the two single mutants On k(cat), indicating cooperativity of Arg-37 and Tyr-103 in promoting catalysis. The extensive activation of the GDP leaving group suggests a mechanism with dissociative character with a cationic oxocarbenium-like transition state and a half-chair conformation of the sugar ring, as found with glycosidase enzymes. Accordingly, Asp-22 which contributes 10(2,1)- to 10(2.6)-fold to k(cat), is positioned to both stabilize a developing cationic center at C I and to accept a hydrogen bond from the C2-OH of the mannosyl group, and His-88, which contributes 10(2.3)-fold to k(cat), is positioned to accept a hydrogen bond from the C3-OH of the mannose facilitating its distortion to a half-chair conformation. Also, the fluorinated substrate GDP-2-fluoro-alpha-D-mannose, for which the oxocarbenium ion-like transition state centered at C1 would be destabilized by electron withdrawal, shows a 16-fold lower k(cat), and a 2.5-fold greater K-m than does GDP-alpha-D-mannose. The product of the contributions to catalysis of Arg-37 and Tyr-103 (taking their cooperativity into account), Arg-52, Arg-65, Mg2+, Asp-22, His-124, and His-88 is >= 10(19), which exceeds the 10, 2 -fold rate acceleration produced by GDPMH by a factor >= 10(7). Hence, additional pairs or groups of catalytic residues must act cooperatively to promote catalysis. (c) 2005 Elsevier B.V. All rights reserved.