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GDP-mannose mannosyl hydrolase (GDPMH) from E. coli catalyzes the hydrolysis of GDP-α- d -sugars to GDP and β- d -sugars by nucleophilic substitution with inversion at the anomeric C1 of the sugar, with general base catalysis by His-124. The 1.3 A 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-NδH⋯Tyr-127-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 kcat and Km. The GDP leaving group is activated by the essential Mg2+ which contributes at least 105-fold to kcat, 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 kcat ranging from 24- to 309-fold. Both Arg-37 and Tyr-103 bind the β-phosphate of the leaving GDP and are only 5.0 A apart. Accordingly, the R37Q/Y103F double mutant shows partially additive effects of the two single mutants on kcat, 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 102.1- to 102.6-fold to kcat, is positioned to both stabilize a developing cationic center at C1 and to accept a hydrogen bond from the C2–OH of the mannosyl group, and His-88, which contributes 102.3-fold to kcat, 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-α- d -mannose, for which the oxocarbenium ion-like transition state centered at C1 would be destabilized by electron withdrawal, shows a 16-fold lower kcat and a 2.5-fold greater Km than does GDP-α- 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 ≥1019, which exceeds the 1012-fold rate acceleration produced by GDPMH by a factor ≥107. Hence, additional pairs or groups of catalytic residues must act cooperatively to promote catalysis.