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 catalyzes the reversible conversion of succinyl-CoA and acetoacetate into acetoacetyl-CoA and succinate through a covalent enzyme thiol ester intermediate, E-CoA, utilizes binding energy from noncovalent interactions with CoA to bring about an increase in kcat/KM of approximately 10(10)-fold. The approximately 40-fold stronger binding of desulfo-CoA (KI = 2.7 +/- 0.7 mM) compared to desulfopantetheine (KI = 110 +/- 15 mM), both of which inhibit competitively with respect to acetoacetyl-CoA, shows that binding to the nucleotide domain of CoA at the active site provides ca. -2.2 kcal/mol of binding energy to stabilize noncovalent complexes with the enzyme. This is much smaller than the ca. -8.9 kcal/mol that the nucleotide domain contributes to the stabilization of the transition state and the ca. -7.2 kcal/mol that it contributes 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 approximately 10(6)-fold increase in kcat/KM that is brought about by binding to this domain is in kcat, which is increased by a factor of about 10(5). Binding to the central pantoic acid domain of CoA is stronger in the transition state than in the Michaelis complex by ca. -3.4 kcal/mol; this corresponds to an additional increase in kcat of approximately 350-fold. Covalent enzyme thiol esters analogous to E-CoA but containing the short-chain CoA analogues N-acetylaletheine (NAA) and N-acetylcysteamine (NAC) are more stable than the enzyme thiol ester containing pantetheine (E-Pant) by approximately 3.5 and approximately 4.8 kcal/mol, respectively. Thus, interactions between the pantoic acid domain of CoA and the active site destabilize E-CoA by approximately 4.8 kcal/mol, approximately 1.3 kcal/mol of which arises from interaction with the amide group of the pantoic acid domain and approximately 3.5 kcal/mol of which arises from interaction with other portions of the pantoic acid domain. E-Pant is more reactive toward acetoacetate and succinate by a factor of approximately 10(7) than E-NAA and E-NAC. This shows that the destabilization caused by these interactions in E-CoA is relieved in the transition state, in which binding to the pantoic acid moiety is strongly favorable with delta delta G approximately -5.2 kcal/mol.(ABSTRACT TRUNCATED AT 400 WORDS)
Whitty
A
A
Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254-9110, USA.
Fierke
C A
CA
Jencks
W P
WP
D000818:Animals; D001665:Binding Sites; D002264:Carboxylic Acids / Q000378:metabolism; D002384:Catalysis; D003065:Coenzyme A / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D015256:Coenzyme A-Transferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006885:Hydroxybutyrates / Q000737:chemistry / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D013438:Sulfhydryl Compounds / Q000378:metabolism / Q000494:pharmacology; D013552:Swine; D013816:Thermodynamics
D002264:Carboxylic Acids; D006885:Hydroxybutyrates; D013438:Sulfhydryl Compounds; C016031:pantoic acid; D015256:Coenzyme A-Transferases; C021460:3-ketoacid CoA-transferase; D003065:Coenzyme A