GDP-mannose hydrolase catalyzes the hydrolysis with inversion of GDP-alpha-D-hexose to GDP and beta-D-hexose by nucleophilic substitution by water at C1 of the sugar. Two new crystal structures (free enzyme and enzyme-substrate complex), NMR, and site-directed mutagenesis data, combined with the structure of the enzyme-product complex reported earlier, suggest a four-stage catalytic cycle. An important loop (L6, residues 119-125) contains a ligand to the essential Mg2+ (Gln-123), the catalytic base (His-124), and three anionic residues. This loop is not ordered in the X-ray structure of the free enzyme due to dynamic disorder, as indicated by the two-dimensional 1H-15N HMQC spectrum, which shows selective exchange broadening of the imidazole nitrogen resonances of His-124 (k(ex) = 6.6 x 10(4) s(-1)). The structure of the enzyme-Mg2+-GDP-mannose substrate complex of the less active Y103F mutant shows loop L6 in an open conformation, while the structure of the enzyme-Mg2+-GDP product complex showed loop L6 in a closed, "active" conformation. 1H-15N HMQC spectra show the imidazole N epsilon of His-124 to be unprotonated, appropriate for general base catalysis. Substituting Mg2+ with the more electrophilic metal ions Mn2+ or Co2+ decreases the pKa in the pH versus kcat rate profiles, showing that deprotonation of a metal-bound water is partially rate-limiting. The H124Q mutation, which decreases kcat 10(3.4)-fold and largely abolishes its pH dependence, is rescued by the Y103F mutation, which increases kcat 23-fold and restores its pH dependence. The structural basis of the rescue is the fact that the Y103F mutation shifts the conformational equilibrium to the open form moving loop L6 out of the active site, thus permitting direct access of the specific base hydroxide from the solvent. In the proposed dissociative transition state, which occurs in the closed, active conformation of the enzyme, the partial negative charge of the GDP leaving group is compensated by the Mg2+, and by the closing of loop L2 that brings Arg-37 closer to the beta-phosphate. The development of a positive charge at mannosyl C1, as the oxocarbenium-like transition state is approached, is compensated by closing the anionic loop, L6, onto the active site, further stabilizing the transition state.