Insights Into The Catalytic Mechanism Of Acetate CoA-transferase By QM/MM Calculation

Acetate CoA-transferase(AarC)belongs to class I CoA transferases that catalyze CoA transfer from acyl-CoA to succinate,which also as an acetic acid resistance factor(aar)in a variant citric acid cycle(CAC)of Acetobacter aceti.The research of the catalytic mechanism of AarC plays a vital role in understanding the process of this variant CAC.In this respect,the researches of the catalytic mechanism of class I CoA transferases has attracted a great deal of attention over the years.In 1967,Jencks et al.put forward the catalytic mechanism of class I Co A-transferases that proceed through two half-reactions of ?PingPong? mechanism with an enzyme-CoA intermediate and proposed a thiol ester linkage to ?-carboxyl group of an enzyme glutamic acid binds the CoA.A few years later,based on the results of molecular exclusion chromatography and kinetic experiment,they suggested that the half-reaction is a stepwise reaction which going through anhydride intermediate.Interestingly,the second half-reaction proceeds in the reverse order of the first half-reaction following exchange of carboxylate product.Until now,the ?PingPong? mechanism of class I CoA-transferases was substantiated by a set of crystal structures.However,these studies are not solve the transition state which exists for a very short time in the reaction of AarC.Over the years,the vigorous development of the theory of chemistry promotes the research of biochemistry process,especially the emergence and development of QM/MM method that provide us a convenient way for theoretically studying the enzyme catalysis mechanism.To elucidate the reaction mechanism of AarC,we carried out molecular dynamic(MD)simulations and quantum mechanical/molecular mechanical(QM/MM)calculations at the atomic level.The results suggest that the step 1 of the half-reaction is accomplished by removing the acyl group from the acyl-CoA to Glu294.The degree of charge transfer and bond distance indicates that the C1-S1 P bond breaks.Meanwhile the OE1-C1 bond forms,resulting in the acetylglutamyl anhydride intermediate.The step 2 of the process is accomplished by the acetate leaving from acetylglutamyl anhydride intermediate to enzyme environment.Mulliken charge redistribution and bond distance indicates that the OE1-CD bond breaks.Meanwhile the CD-S1 P bond forms,resulting in the glutamyl-CoA thioester product.In AarC reaction,the rate-limiting step is step 1 with the energy barrier of 14.89 kcal/mol in first half-reaction.In addition,in AarC mutant N347 A,the energy barrier of rate-limiting step is 18.50 kcal/mol,which should be the function of Asn347 vanish that is consistent with previous experimental proposal.The results suggest that residue Asn347 has double roles in promoting the half-reaction of AarC.On the one hand,Asn347 helps enzyme to stabilize the structure of transition state and intermediate.On the other hand,Asn347 anchors the side chain of Glu294 in suitable orientation and restricts the motion of Glu294.In the process of catalytic mechanism of half-reaction,we also propose several residues including Val270,Gly388,and Ser389,which play vital roles in helping enzyme to stabilize the structure of transition state and intermediate.The study presented here opens up our horizon of the catalytic mechanism of AarC,especially provides crucial information in studying the action mechanism of variant citric acid cycle of Acetobacter aceti.