Magnetic resonance and kinetic studies on the manganese activated arginine kinase reaction.

Abstract

Proton relaxation rate studies have been used to study the interaction between arginine kinase from two sources, Homarus vulgaris and Homarus americanus, and manganous ion, MnADP‐ and MnATP2‐, respectively. There was negligible interaction between Mn2+ and the H. vulgaris enzyme but a small effect with the H. americanus enzyme. In both cases, a large enhancement of the proton relaxation rate was seen on the addition of ADP or ATP to a solution containing Mn2+ and enzyme. It was concluded that the enzyme combines with MnADP− or MnATP2‐ rather than with Mn2+.Enhancements of the proton relaxation rate of the ternary complexes, MnADP‐E (45), Mn2′‐dADP‐E (28) and MnATP‐E (17), were obtained with the H. vulgaris arginine kinase and Mn3′‐dADP− and Mn2′‐dATP2‐ were also demonstrated to form ternary complexes with this enzyme. For arginine kinase from H. americanus, values for MnADP‐E (17), Mn2′‐dADP‐E (approx. 12) and MnATP‐E (approx. 8) were determined. A comparative kinetic study demonstrated reasonable agreement between the kinetically determined Km values and dissociation constants from the proton relaxation rate data. It was also found that for both enzymes, there was a correlation between the enhancement and maximum velocity values, ADP > 2′‐dADP and ATP > 2′‐dATP which could be interpreted as indicative of a graded degree of substrate induced conformational change. The proton relaxation rate and kinetic studies indicated small but probably significant differences in the properties of the two enzymes.Proton relaxation rate studies provided evidence for a second substrate induced conformational change on the addition of l‐arginine to the ternary MnADP‐enzyme complex. No significant effect was observed with d‐arginine, which is a competitive inhibitor with respect to l‐arginine.