Studies on adenosine triphosphate transphosphorylases: XIII. Kinetic properties of the crystalline rabbit muscle ATP-AMP transphosphorylase (adenylate kinase) and a comparison with the crystalline calf muscle and liver adenylate kinases

Abstract

The steady-state kinetics, at pH 7.4, 25 °C and essentially fixed (Γ/2) of 0.16–0.18, of the rabbit muscle, calf muscle, and calf liver adenylate kinases all seem to be adequately expressed by a random quasi-equilibrium type of mechanism with a rate-limiting step largely at the interconversion of the ternary complexes. However, the data presented, by themselves, will not exclude the possibility of a random mechanism in which product release is rate limiting. In the case of the rabbit muscle enzyme, five substrate pairs for the forward reaction (MgATP 2−/AMP 2−; MgdATP 2−/dAMP 2−; MgATP 2−/dAMP 2−; MgdATP 2−/AMP 2−; MgϵATP 2−/AMP 2−) and two substrate pairs for the reverse reaction (MgADP −/ADP 3−; MgdADP −/DP 3−) were studied under conditions where a careful and systematic control was exercised over the metal complex nucleotide species. For the forward reactions between MgATP 2− + AMP 2−, and MgdATP 2− + dAMP 2−, the estimated values for K ̄ s,1 and K s,1 (i.e., intrinsic dissociation constants of the substrate from the binary and ternary complexes) differed, pointing to substrate (s) induced conformational changes in the ternary complexes. The fact that similar derived values either for K ̈ AMP 2− - and for K AMP 2− could be estimated for each of the above three sets of substrate pairs in the forward reaction, supports the conclusion that a common rate-limiting step exists and is likely in the interconversion of the ternary complexes. ϵAMP 2− was found to be a competitive inhibitor of AMP 2− (at fixed MgATP 2−) and MgdADP − to be noncompetitive inhibitor of ADP 3− (at fixed MgADP −), in support of the conclusion of D. G. Rhoads and J. M. Lowenstein ( J. Biol. Chem. (1968) 243, 3963–3972) that a separate site exists for the magnesium chelates of the nucleotide substrates (MgATP 2− and MgADP −) and for the uncomplexed nucleotide substrates (ADP 3− and AMP 2−). Distinguishing kinetic features of the calf liver adenylate kinase over the calf muscle isoenzyme are: 1. ▪ (3) inhibition of the liver enzyme by phosphoenolpyruvate; and 2. (4) relatively weak inhibition by P 1,P 5-di-(adenosine-5′) pentaphosphate of the liver enzyme compared to the muscle enzyme (S. A. Kuby et al. (1978) Arch. Biochem. Biophys. 187, 34–52). A thermodynamic value for K eq = (MgADP −) (ADP 3−) ( MgATP 2−) (AMP 2−) = 2.7 (±0.6) × 10 −1 has been arrived at, which compared favorably with the average value from its kinetic estimation via four Haldane relations for the three adenylate kinase sets of data.