Magnetic Resonance and Kinetic Studies Related to the Manganese Activation of the Adenylate Kinase Reaction

Proton relaxation rate (PRR) studies indicated that there was little direct interaction between manganous ion and adenylate kinase. However, a large enhancement of the PRR of water was observed in the presence of ATP; for the enzyme combined with manganous-ATP complex rather than directly with the metal ion. Thus adenylate kinase is similar to creatine kinase but not to pyruvate kinase, with respect to the formation of the ternary enzyme-metal-substrate complex. The enhancement of the PRR of the ternary complex, MnATP-adenylate kinase, was determined to be 35 at 25 Mc per sec, pH 8.0, and 24°. The dissociation constant of MnATP2- from the ternary complex was determined to be 0.5 x 10-4 m from the PRR data. A combination of PRR measurements and of free manganese determinations by electron paramagnetic resonance was used to determine the dissociation constant for MnATP2- and also for the free nucleotide from the enzyme. Values of 0.9 x 10-4 m and 0.3 x 10-4 m, respectively, were obtained. These agree reasonably with previous results from kinetic and thermodynamic data. The enhancement factors, et, of the PRR of the ternary complexes of adenylate kinase with the manganese complexes of a number of nucleoside triphosphates were determined. The order of et was found to be: MnATP (35) g Mn-2'-dATP (20) g MnGTP (8) g MnITP (5) g MnCTP (4) and MnUTP (4) at 24°. Also, a value of 8 for the ternary complex formed between adenylate kinase, manganous ion, and tripolyphosphate was obtained. A comparative kinetic study was carried out with these compounds with manganous ion as activator. For the purine nucleotides the order of maximum velocities was ATP g 2'-dATP g GTP g ITP. The correlation between et and the maximum velocities could be interpreted in part as indicative of some graded degree of conformational change induced in the protein by these substrates. This correlation could not be extended to the pyrimidine nucleotides, and tripolyphosphate did not give a measurable velocity. A temperature study of the variation of the PRR for the ternary complexes formed with adenylate kinase and MnATP2- and Mn-2'-dATP2- was carried out over the range 2–42°. The PRR increased with increasing temperature for both complexes, which is characteristic of a chemical exchange-dominated process.