Investigations of anion binding sites in transition state analogue complexes of creatine kinase by infrared spectroscopy.

A specific class of anions inhibit creatine kinase by stabilizing the dead-end complex, enzymedivalent cation*ADP*creatine (Watts, D. C. (1973) The Enzymes 8, 383-455). The inhibitory action o? the anions is attributed to the ability of the anions to mimic the equatorial PO3 plane formed by the migrating phosphoryl group in the transition state of the reaction. Infrared spectroscopy has been used to investigate the mode of binding of the inhibitory anions, thiocyanate, azide, and nitrate to the dead-end complex of creatine kinase. The infrared absorptions for these anions undergo characteristic changes in frequency or in multiplicity when the anions are liganded to various divalent cations, and infrared spectra for the enzymic complexes with the anions provide a means for recognizing anionmetal ion coordination at the active site of creatine kinase. Infrared spectra for the complexes of the anions with enzyme l divalent cation. ADP. creatine species coincide with the vibrational spectra obtained for the simple anion*metal ion complexes in concentrated solutions of the metal-anion salts. These cation specific responses of the infrared absorptions for the enzymebound anions provide cogent evidence for liganding of the anions to the activating divalent cation. Since none of the anions investigated exhibit a high affinity for the cations, Mg(II), Mn(II), and Co(I1) in free solution, promotion of anion-metal ion coordination in the dead-end complex of creatine kinase connotes coordination of the metal ion to an oxygen atom of the equatorial POs plane in the transition state of the reaction. Such a coordination scheme would be compatible with an inline displacement mechanism for the reaction where the divalent cation activates through template, polarization, and charge shielding influences.