The intrinsic reactivity of ATP and the catalytic proficiencies of kinases acting on glucose, N-acetylgalactosamine, and homoserine: a thermodynamic analysis.

To evaluate the rate enhancements produced by representative kinases and their thermodynamic basis, rate constants were determined as a function of changing temperature for 1) the spontaneous methanolysis of ATP and 2) reactions catalyzed by kinases to which different mechanisms of action have been ascribed. For each of these enzymes, the minor effects of changing viscosity indicate that k(cat)/K(m) is governed by the central chemical events in the enzyme-substrate complex rather than by enzyme-substrate encounter. Individual Arrhenius plots, obtained at intervals between pH 4.8 and 11.0, yielded Delta H(#) and T Delta S(#) for the nonenzymatic methanolysis of ATP(2-), ATP(3-), and ATP(4-) in the absence of Mg(2+). The addition of Mg(2+) led to partly compensating changes in Delta H(#) and T Delta S(#), accelerating the nonenzymatic methanolysis of ATP 11-fold at pH 7 and 25 degrees C. The rate enhancements produced by yeast hexokinase, homoserine kinase, and N-acetylgalactosamine kinase (obtained by comparison of their k(cat)/K(m) values in the presence of saturating phosphoryl acceptor with the second order rate constant for methanolysis of MgATP) ranged between 10(12)- and 10(14)-fold. Their nominal affinities for the altered substrates in the transition state were 2.1 x 10(-16) m for N-acetylgalactosamine kinase, 7.4 x 10(-17) m for homoserine kinase, and 6.4 x 10(-18) m for hexokinase. Compared with nonenzymatic phosphoryl transfer, all three kinases were found to produce major reductions in the entropy of activation, in accord with the likelihood that substrate juxtaposition and desolvation play prominent roles in their catalytic action.