Role of Binding Energy with Coenzyme A in Catalysis by 3-Oxoacid Coenzyme A Transferase

• Published in: Biochemistry (Easton) 1995-09, Vol.34 (37), p.11678-11689
• Main Authors: Whitty, Adrian, Fierke, Carol A, Jencks, William P
• Format: Article
• Language: English
• Subjects: Animals; Binding Sites; Carboxylic Acids - metabolism; Catalysis; Coenzyme A - chemistry; Coenzyme A - metabolism; Coenzyme A - pharmacology; Coenzyme A-Transferases - antagonists & inhibitors; Coenzyme A-Transferases - metabolism; Hydroxybutyrates - chemistry; Hydroxybutyrates - metabolism; In Vitro Techniques; Kinetics; Sulfhydryl Compounds - metabolism; Sulfhydryl Compounds - pharmacology; Swine; Thermodynamics
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00037a005

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.