| Abstract
| - S-Adenosylhomocysteine hydrolase (AdoHcy hydrolase) crystallizes from solutions containingthe intermediate analogue neplanocin A with the analogue bound in its 3‘-keto form at the active sites ofall of its four subunits and the four tightly bound cofactors in their reduced (NADH) state. The enzymeis in the closed conformation, which corresponds to the structure in which the catalytic chemistry occurs.Examination of the structure in the light of available, very detailed kinetic studies [Porter, D. J., Boyd, F.L. (1991) J. Biol. Chem.266, 21616−21625. Porter, D. J., Boyd, F. L. (1992) J. Biol. Chem.267, 3205−3213. Porter, D. J. (1998) J. Biol. Chem.268, 66−73] suggests elements of the catalytic strategy ofAdoHcy hydrolase for acceleration of the reversible conversion of AdoHcy to adenosine (Ado) andhomocysteine (Hcy). The enzyme, each subunit of which possesses a substrate-binding domain that inthe absence of substrate is in rapid motion relative to the tetrameric core of the enzyme, first binds substrateand ceases motion. Probably concurrently with oxidation of the substrate to its 3‘-keto form, the closedactive site is “sealed off” from the environment, as indicated by a large (108-9-fold) reduction in the rateof departure of ligands, a feature that prevents exposure of the labile 3‘-keto intermediates to the aqueousenvironment. Elimination of the 5‘-substituent (Hcy in the hydrolytic direction, water in the syntheticdirection) generates the central intermediate 4‘,5‘-didehydro-5‘-deoxy-3‘-ketoadenosine. Abortive 3‘-reduction of the central intermediate is prevented by a temporary suspension of all or part of the redoxcatalytic power of the enzyme during the existence of the central intermediate. The abortive reduction is104-fold slower than the productive reductions at the ends of the catalytic cycle and has a rate constantsimilar to those of nonenzymic intramolecular model reactions. The mechanism for suspending the redoxcatalytic power appears to be a conformationally induced increase in the distance across which hydridetransfer must occur between cofactor and substrate, the responsible conformational change again beingthat which “seals” the active site. The crystal structure reveals a well-defined chain of three water moleculesleading from the active site to the subunit surface, which may serve as a relay for proton exchange betweensolvent and active site in the closed form of the enzyme, permitting maintenance of active-site functionalgroups in catalytically suitable protonation states.
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