ROLE OF BINDING-ENERGY WITH COENZYME-A IN CATALYSIS BY 3-OXOACID COENZYME-A TRANSFERASE

Succinyl-CoA:3-oxoacid coenzyme A transferase (EC 2.8.3.5), which cata lyzes the reversible conversion of succinyl-CoA and acetoacetate into acetoacetyl-CoA and succinate through a covalent enzyme thiol ester in termediate, E-CoA, utilizes binding energy from noncovalent interactio ns with CoA to bring about an increase in k(cat)/K-M of approximate to 10(10)-fold. The approximate to 40-fold stronger binding of desulfo-C oA (K-I = 2.7 +/- 0.7 mM) compared to desulfopantetheine (K-I = 110 +/ - 15 mM), both of which inhibit competitively with respect to acetoace tyl-CoA, shows that binding to the nucleotide domain of CoA at the act ive site provides ca. -2.2 kcal/mol of binding energy to stabilize non covalent complexes with the enzyme. This is much smaller than the ca. -8.9 kcal/mol that the nucleotide domain contributes to the stabilizat ion of the transition state and the ca. -7.2 kcal/mol that it contribu tes to stabilizing the E-CoA intermediate [Fierke, C. A., & Jencks, W. P. (1986) J. Biol. Chem. 261, 7603-7606]. This shows that most of the approximate to 10(6)-fold increase in k(cat)/K-M that is brought abou t by binding to this domain is in k(cat), which is increased by a fact or of about 10(5). Binding to the central pantoic acid domain of CoA i s stronger in the transition state than in the Michaelis complex by ca . -3.4 kcal/mol; this corresponds to an additional increase in k(cat) of approximate to 350-fold. Covalent enzyme thiol esters analogous to E-CoA but containing the short-chain CoA analogues N-acetylaletheine ( NAA) and N-acetylcysteamine (NAG) are more stable than the enzyme thio l ester containing pantetheine (E-Pant) by approximate to 3.5 and appr oximate to 4.8 kcal/mol, respectively. Thus, interactions between the pantoic acid domain of CoA and the active site destabilize E-CoA by ap proximate to 4.8 kcal/mol, approximate to 1.3 kcal/mol of which arises from interaction with the amide group of the pantoic acid domain and approximate to 3.5 kcal/mol of which arises from interaction with othe r portions of the pantoic acid domain. E-Pant is more reactive toward acetoacetate and succinate by a factor of approximate to 10(7) than E- NAA and E-NAC. This shows that the destabilization caused by these int eractions in E-CoA is relieved in the transition state, in which bindi ng to the pantoic acid moiety is strongly favorable with Delta Delta G approximate to-5.2 kcal/mol. Thus, interactions with the nucleotide a nd pantoic acid domains of CoA play distinct roles in catalysis by CoA transferase: Binding to the nucleotide domain accelerates the formati on of E-CoA and stabilizes it relative to the Michaelis complex, but d oes not significantly contribute to catalysis of the second half-react ion. Binding to the pantoic acid domain also contributes to accelerati ng the formation of E-CoA; however, binding to this domain destabilize s the E-CoA intermediate by approximate to 4.8 kcal/mol. Relief of thi s destabilization, coupled with strongly favorable binding to the pant oic acid domain in the transition state, lowers the activation barrier for the second half-reaction by approximate to 10 kcal/mol, which cor responds to an increase in k(cat) for the reaction of E-CoA with aceto acetate or succinate by a factor of approximate to 10(7). This large a nd critical change in the binding interactions between the enzyme and CoA involves only the small region of the CoA molecule containing the alpha, beta, and gamma carbon atoms of the pantoic acid domain and the ir substituents.