Potential of mean force calculation for the proton and hydride transfer reactions catalyzed by medium-chain acyl-CoA dehydrogenase: effect of mutations on enzyme catalysis.
Potential of mean force calculations have been performed on the wild-type medium-chain acyl-CoA dehydrogenase (MCAD) and two of its mutant forms. Initial simulation and analysis of the active site of the enzyme reveal that an arginine residue (Arg256), conserved in the substrate-binding domain of this group of enzymes, exists in two alternate conformations, only one of which makes the enzyme active. This active conformation was used in subsequent computations of the enzymatic reactions. It is known that the catalytic alpha,beta-dehydrogenation of fatty acyl-CoAs consists of two C-H bond dissociation processes: a proton abstraction and a hydride transfer. Energy profiles of the two reaction steps in the wild-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 approximately 2 kcal/mol, indicating that both may contribute to the rate-limiting process. Thus this may be a stepwise dissociation mechanism whose relative barriers can be tuned by suitable alterations of the substrate and/or enzyme. Analysis of the structures along the reaction path reveals that Arg256 plays a key role in maintaining the reaction center hydrogen-bonding network involving the thioester carbonyl group, which stabilizes transition states as well as the intervening transient species. Mutation of this arginine residue to glutamine increases the activation barrier of the hydride transfer reaction by approximately 5 kcal/mol, and the present simulations predict a substantial loss of catalytic activity for this mutant. Structural analysis of this mutant reveals that the orientation of the thioester moiety of the substrate has been changed significantly as compared to that in the wild-type enzyme. In contrast, simulation of the active site of the Thr168Ala mutant shows no significant change in the relative orientation of the substrate and the cofactor in the active site; as a result, this mutation has very little effect on the overall reaction barrier, and this is consistent with the experimental data. This study demonstrates 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.
Bhattacharyya
Sudeep
S
Department of Chemistry, University of Minnesota, Smith Hall, 207 Pleasant Street SE, Minneapolis, Minnesota 55455-0431, USA. sudeep@chem.umn.edu
Ma
Shuhua
S
Stankovich
Marian T
MT
Truhlar
Donald G
DG
Gao
Jiali
J
D042964:Acyl-CoA Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000215:Acylation; D000818:Animals; D001120:Arginine / Q000378:metabolism; D002384:Catalysis; D006224:Cricetinae; D004789:Enzyme Activation; D006860:Hydrogen Bonding; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D011522:Protons; D013379:Substrate Specificity
D011522:Protons; D001120:Arginine; D042964:Acyl-CoA Dehydrogenase