Magnetic Resonance Investigations of the Metal Complexes Formed in the Manganese-activated Creatine Kinase Reaction
W. J. O'Sullivan 1 and Mildred Cohn 1
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1 From the Johnson Research Foundation, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
Stability constants for the manganese complexes of various components of the creatine kinase reaction, including possible buffers, have been determined by electron paramagnetic resonance (EPR). EPR and proton relaxation rate (PRR) studies indicated that such interaction as occurred between manganese and creatine kinase was nonspecific and relatively weak, unlike the interaction between manganese and pyruvate kinase (12). The maximum enhancement of the PRR induced by binding of manganese to the enzyme was estimated to be 1.5, and this was considerably reduced at high ionic strength.
Enhancements of the PRR of 19.4 at 25 mc per sec for the ternary complex, creatine kinase-manganese-adenosine diphosphate, at pH 8.0 and 24° and of 9.8 at 25 mc per sec for creatine kinase-manganese-ATP at pH 7.5 and 24° were obtained. Values of the dissociation constants of the metal nucleotides from the ternary complexes were obtained with the use of PRR data alone or by a combination of PRR and EPR data. It was also possible to obtain estimates of the dissociation constants of the free nucleotides from their binary complexes with the enzyme.
The values for the dissociation constants were in satisfactory agreement with those derived from a limited study of the activation of creatine kinase by manganese, consistent with the enzymatic reaction obeying equilibrium kinetics. The values agreed with the results obtained by J. F. Morrison and M. L. Uhr (personal communication) for the manganese-activated reaction and were similar to the dissociation constants obtained previously from kinetic and thermodynamic data with magnesium as the activating ion. It was concluded that the equilibria measured by magnetic resonance methods corresponded to the equilibria among the kinetically active species.
Pyrophosphate and tripolyphosphate also formed ternary complexes with low values of 
t 2.0 and 2.5, respectively. No evidence could be found for the formation of ternary complexes with AMP, inorganic phosphate, phosphocreatine or creatine. However, evidence for the formation of the abortive quaternary complex, manganese-ADP-creatine kinase-creatine, was obtained. The enhancement, q, of the quaternary complex was less than t under the conditions measured. The difference between t and q was found to increase as the temperature decreased, since the PRR of the ternary complex had a negative temperature coefficient and the PRR of the quaternary complex had a positive temperature coefficient.
From the magnitude of the relaxation rates of the ternary and quaternary complexes and the magnitude and signs of the temperature coefficients, approximate calculations indicated that the large enhancement of PRR in these complexes results predominantly from the fact that the rotational motion of water in the coordination sphere of the metal ion is greatly hindered in both complexes. On the other hand, the decrease in the magnitude of the PRR between the ternary and quaternary complexes appears to be caused by a decrease in the rate of water exchange between the metal coordination sphere and the solvent for the quaternary complex.
Submitted on January 10, 1966
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