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Nucleotide Specificity and Conformation of the Active Site of Creatine Kinase

MAGNETIC RESONANCE AND SULFHYDRYL REACTIVITY STUDIES

From the ¹ From the Johnson Research Foundation, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

The manganese-enzyme-substrate complexes of a number of nucleoside diphosphate substrates of creatine kinase showed a range of enhanced values of the longitudinal proton relaxation rate of water with respect to the manganous aquocation, although the small enhancement factors, _a, of all the binary manganese-nucleotide complexes were the same, approximately 1.6. The order of the values of _t, the enhancement factor of the respective ternary enzyme-metal-substrate complexes, was: manganese-adenosine diphosphate (19.4) > Mn-3'-dADP (7.7) > Mn-2'-dADP (6.7) > manganese-inosine diphosphate (5) > manganese-guanosine diphosphate (4). This same order is observed for the maximum velocity of the enzymatic reaction with these substrates. There was no significant difference in the dissociation constants of the enzyme-metal-substrate complexes for the three manganese-adenine nucleotides. Values of _t for the nucleoside triphosphates were: MnATP (9.8) > Mn-2'-dATP (4.5) > MnGTP (3.7) > MnITP (3.2). The order of relative velocities was found to be MnATP > Mn-2'-dATP > MnITP > MnGTP.

The relative effects of a number of substrates and inhibitors of creatine kinase on the rate of reaction between iodoacetic acid and the essential sulfhydryl groups of the enzyme was also investigated. Metal nucleoside diphosphates increased the —SH reactivity in the order M-ADP > M-2'-dADP > M-IDP > M-GDP, while the free nucleotides protected in the order ADP > 2'-dADP > IDP > GDP. Both the metal complexes and the free forms of nucleoside triphosphates protected creatine kinase against iodoacetic acid, although the effects were much greater for the free nucleotides. With both series of compounds, an order similar to that for the diphosphates was observed.

The parallel order of the values of _t, the maximum velocities of the enzymatic reaction, and the rate constants for the iodoacetic acid reaction with the different metal-nucleotides may be ascribed to differing degrees of conformational change induced at the active site of creatine kinase. Thus, the properties of water in the coordination sphere of the manganese in the enzyme-metal-substrate complex as manifested by the enhancement of proton relaxation rate may be used as a probe to reveal changes in the conformation of the enzyme at its active site in the same way as the modification of the reactivity of the essential cysteine residues is used. With the latter criterion, the effect of binding free nucleotides and metal-nucleotides could be investigated and the observed rate constants for the reaction between iodoacetic acid and creatine kinase could be explained by induced conformational changes, which increased the accessibility of the —SH groups upon binding of metal nucleotides and decreased the accessibility of the —SH groups upon binding of the free nucleotides.

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