| Abstract
| - Domain motions of S-adenosyl-l-homocysteine (AdoHcy) hydrolase have been detected bytime-resolved fluorescence anisotropy measurements. Time constants for reorientational motions in thenative enzyme were compared with those for enzymes where key residues were altered by site-directedmutation. Mutations M351P, H353A, and P354A were selected in a hinge region for motion between theopen and closed forms of the enzyme, as identified in a previous normal-mode study [Wang et al. (2005)Domain motions and the open-to-closed conformational transition of an enzyme: A normal-mode analysisof S-adenosyl-l-homocysteine hydrolase, Biochemistry44, 7228−7239]. In wild-type, substrate-freeAdoHcy hydrolase (NAD+ cofactor in each subunit), reorientational motions were detected on time scalesof 10−20 and 80−90 ns. The faster motion is attributed to the domain motion, and the slower motion isattributed to the tumbling of the enzyme. The domain motion was also detected for the enzyme complexesE(NADH/3‘-keto-adenosine) and E(NAD+/3‘-deoxyadenosine) but was absent for the complex E(NADH/3‘-keto-neplanocin A). The results indicate that AdoHcy hydrolase exists in equilibrium of open and closedstructures, with the equilibrium shifted toward the more mobile open form for the substrate-free enzyme,E(NAD+), and for intermediates formed early in the catalytic cycle after substrate binding or formed lateprior to product release, E(NAD+/ligand). However, the strong inhibitor neplanocin A upon bindingundergoes oxidation, forming the complex E(NADH/3‘-keto-neplanocin). For this complex, which isanalogous to the enzyme complex with the central catalytic intermediate, the equilibrium was shiftedtoward the more rigid closed form. A similar pattern was observed for M351P and P354A mutants. Incontrast, the domain motion could not be detected, either in the absence or presence of ligands or withthe cofactor in either the oxidized or reduced state, for the H353A protein, suggesting that this mutationchanges the hinge-bending dynamics of the enzyme.
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