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Abstract
CoA transferase is inactivated by the thiols, N-acetylcysteamine, N-acetylaletheine and methyl mercaptopropionate. The mechanism of inactivation is concluded to be: (UNFORMATTED TABLE FOLLOWS)
O O
RSH + E-CSCoA E-CSR + CoASH
(inactive)
(TABLE ENDS)
The rate and equilibrium constants for the reaction of these enzyme thioesters were measured with water, thiols and carboxylate substrates.
Pantetheine reacts with E-MMP to form and E-Pantetheine thioester, which hydrolyzes rapidly (k(,hyd) = 2 min('-1)) to form E-COO('-). The reactivity of E-Pantetheine with carboxylate substrates and thiols was measured. E-Pantetheine is 5.4 kcal/mol less stable than E-CoA, but it is as reactive as E-CoA.
Kinetic constants for turnover were measured for the substrates, S-acetoacetyl-pantetheine, S-acetoacetyl-deamino-CoA and S-acetoacetyl-dephospho-CoA.
These studies with CoA fragments demonstrated that: (a) CoA transferase utilizes 18 kcal/mol intrinsic CoA binding energy to catalyze the reaction of acacCoA with enzyme, and utilizes 11 kcal/mol of CoA binding energy for catalysis of the reaction of E-CoA with acetoacetate. (b) This utilization of CoA binding energy is split into two functional moieties. Intrinsic binding energy of pantetheine is utilized to provide almost all of the CoA-induced rate acceleration while the 3'-P,5'-ADP moiety provides expressed binding energy to stabilize the E-Sr intermediate. (c) Interactions with the pantoic acid group of pantetheine are responsible for the majority of the rate acceleration. These provide 4.5 kcal/mol transition state stabilization and 5.4 kcal/mol ground state destabilization. (d) The entire CoA molecule must be covalently joined to observe the CoA-induced rate acceleration.
The buffer dependence of the rate of a proton transfer reaction from an acid on fumarase (pK(,a) = 6.3) to solvent was assayed using ('14)C/('3)H exhange rate ratios and compared to the buffer dependence of a nonenzymic proton transfer. Under conditions where the non- enzymic proton transfer reaction is catalyzed by a variety of buffers (i.e., tris, phosphate, acetate), the enzymic proton transfer shows little or no catalysis. The estimated rate constant for the enzymic proton transfer is fast, 10('6)-10('7) s('-1). The absence of buffer catalysis requires that the fumarase acid is shielded from the solvent so buffer bases cannot diffuse up to it.
The reaction of fumarase with fluorofumarate was shown to occur in three steps; enzyme-catalyzed formation of fluoromalate, rapid elimination of hydrofluoric acid to form keto-oxaloacetate (k > 0.2 s('-1)), followed by buffer-catalyzed enolization. (Abstract shortened with permission of author.)
