Structure of metal-nucleotide complexes bound to creatine kinase: phosphorus-31 NMR measurements using manganese(II) and cobalt(II)

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Abstract
The structures of metal-nucleotide complexes bound to rabbit muscle creatine kinase were studied by making measurements of paramagnetic effects of 2 dissimilar activating paramagnetic cations, Mn(II) and Co(II), on the spin-relaxation rates of the 31P nuclei of ATP and ADP in these complexes. The experiments were performed on enzyme-bound complexes, thereby limiting the contributions to the observed relaxation rates to 2 exchanging complexes (with and without the cation). Measurements were made as a function of temperature in the range 5-35.degree. C and at 3 31P NMR frequencies, 81, 121.5 and 190.2 MHz, in order to determine the effect of exchange on the observed relaxation rates. The relaxation rates in E .cntdot. MnADP and E .cntdot. MnATP are independent of frequency, and their temperature variation yields activation energies (.DELTA.E) in the range 5-8 kcal/mol; in the transition-state analogue complex E .cntdot. MnADP .cntdot. NO3- .cntdot. Cre (Cre is creatine), .DELTA.E is increased to 17.3 kcal/mol. The relaxation rates in the Mn(II) complexes apparently are exchange limited and are incapable of providing structural data. Use of line-width measurements to estimate the lifetime of the paramagnetic complex evidently leads to incorrect results. The relaxation rates in E .cntdot. CoADP and E .cntdot. CoATP exhibit frequency dependence and .DELTA.E values in the range 1-3 kcal/mol, i.e., these rates depend on the Co(II)-31P distances, whereas those in the E .cntdot. CoADP .cntdot. NO3- .cntdot. Cre complex have .DELTA.E .apprx. 18 kcal/mol and are significantly contributed by exchange. Difficulties involved in estimating the electron relaxation times in E .cntdot. CoADP and E .cntdot. CoATP restrict the calculation of Co(II)-31P distances in these complexes to lower and upper limits. These distances were all in the range 2.4-4.3 .ANG., similar to those for free complexes and appropriate for direct coordination between Co(II) and the phosphate groups. This conclusion is in agreement with that reached by using Mn(II) EPR on this enzyme [Leyh, T. S., Goodhart, P. J., Nguyen, A. C., Kenyon, G. L., and Reed, G. H. (1985)] and is in contrast with the conclusion of 2nd-hydration-sphere coordination for Mn(II)-nucleotide complexes of pyruvate kinase [Sloan, D. L., and Mildvan, A. S.] reached by using 31P relaxation measurements.