Studies on Adenosine Triphosphate Transphosphorylases IX. KINETIC PROPERTIES OF THE CRYSTALLINE ADENOSINE TRIPHOSPHATE-CREATINE TRANSPHOSPHORYLASE FROM CALF BRAIN

Abstract The steady state kinetics of the calf brain ATP-creatine transphosphorylase seems to be adequately expressed by a random quasi-equilibrium type of mechanism with a rate-limiting step at the interconversion of the ternary complexes and for a case without independent binding of the substrates. Values for the kinetic parameters at pH 8.8, 30°, have been deduced. The over-all equilibrium constant, calculated kinetically from the Haldane relations, agreed satisfactorily with the thermodynamic value assigned previously (Kuby and Noltmann, The enzymes, Vol. 6, 1962, p. 515). Values for Ks1 versus Ks1 (i.e. intrinsic dissociation constants of the substrate from the ternary and binary complexes, respectively) differed very significantly (e.g. for the forward reaction: Kmgatp2- and Kmgatp2- were 1.35 x 10-4 and 0.93 x 10-3 m, Kcreatine and Kcreatine were 3.7 x 10-3 m and 2.9 x 10-2 m, respectively). The possibility might then be entertained, that at pH 8.8, an enhancement in binding of the individual substrate in the ternary complex has occurred compared to the binary enzyme-substrate complex (cf. Morrison and James, Biochem. J., 97, 37 (1965)). Also, it has been tentatively concluded that ADP3- may compete with MgADP- for binding to the enzyme with a type of inhibition at high ADP0 concentrations which has been evaluated in terms of an abortive and inactive ternary complex with creatine phosphate2-, and which forms also in a random manner. The kinetic parameters have also been estimated for the calf muscle isoenzyme at pH 8.8 (and calculated for the above mechanism). Certain distinguishing kinetic features of each isoenzyme at pH 8.8 are then briefly outlined. A further comparison of the calf brain enzyme's kinetic data obtained at pH 8.8 with those obtained at pH 7.4 reveal several significant differences in the derived parameters, especially in Vmaxreverse (with a large increase), in Kcr∼p2- (with a large decrease), and in comparatively smaller (or even insignificant) decreases between respective Ks1 and Ks1 values (in which case, the brain-type enzyme seems to approach the muscle-type enzyme in its kinetic characteristics). An over-all evaluation of the data (physical, chemical, and kinetic) gathered for the calf brain ATP-creatine transphosphorylase seems to lead to the conclusion that it is susceptible to gross conformational changes as a result of environmental influences, in contrast to the more stable molecular unit to be found in the muscle-type ATP-creatine transphosphorylase.