| Abstract
| - Potential of mean force calculations have been performed on the wild-type medium-chainacyl-CoA dehydrogenase (MCAD) and two of its mutant forms. Initial simulation and analysis of theactive site of the enzyme reveal that an arginine residue (Arg256), conserved in the substrate-bindingdomain of this group of enzymes, exists in two alternate conformations, only one of which makes theenzyme active. This active conformation was used in subsequent computations of the enzymatic reactions.It is known that the catalytic α,β-dehydrogenation of fatty acyl-CoAs consists of two C−H bond dissociationprocesses: a proton abstraction and a hydride transfer. Energy profiles of the two reaction steps in thewild-type MCAD demonstrate that the reaction proceeds by a stepwise mechanism with a transient species.The activation barriers of the two steps differ by only ∼2 kcal/mol, indicating that both may contributeto the rate-limiting process. Thus this may be a stepwise dissociation mechanism whose relative barrierscan be tuned by suitable alterations of the substrate and/or enzyme. Analysis of the structures along thereaction path reveals that Arg256 plays a key role in maintaining the reaction center hydrogen-bondingnetwork involving the thioester carbonyl group, which stabilizes transition states as well as the interveningtransient species. Mutation of this arginine residue to glutamine increases the activation barrier of thehydride transfer reaction by ∼5 kcal/mol, and the present simulations predict a substantial loss of catalyticactivity for this mutant. Structural analysis of this mutant reveals that the orientation of the thioestermoiety of the substrate has been changed significantly as compared to that in the wild-type enzyme. Incontrast, simulation of the active site of the Thr168Ala mutant shows no significant change in the relativeorientation of the substrate and the cofactor in the active site; as a result, this mutation has very littleeffect on the overall reaction barrier, and this is consistent with the experimental data. This studydemonstrates that significant insights into the catalytic mechanism can be obtained from simulation studies,and the results can be used to design novel mechanistic probes for the enzyme.
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