Magnetic resonance studies of three forms of creatine kinase. Comparison of the properties of native, CH-S-blocked, and H2NCOCH-blocked enzymes.

Abstract

Magnetic resonance methods are applied in a comparative study of native creatine kinase from rabbit muscle with two sulfhydryl-modified forms of the enzyme--one inactive form obtained by reaction of the enzyme with iodoacetamide and one form with reduced activity obtained by reaction of the iodoacetamide-sensitive sulfhydryl group with methyl methanethiolsulfonate, which blocks the sulfhydryl with a CH3S-group. Water proton relaxation rate (PRR) titrations with the CH3S-blocked enzyme show that the modification does not alter appreciably the affinities of the enzyme for MnADP and for creatine in the presence of MnADP. Similar measurements for the H2NCOCH2-blocked enzyme indicate that this modification weakens the affinity of the enzyme for MnADP. In agreement with previous findings, there is no observable change in the PRR enhancement upon additions of creatine to solutions of the ternary complex, enzyme-MnADP, for the H2NCOCH2-blocked enzyme. PRR titrations enabled the measurement of binding of creatine to the ternary CH3S-enzyme-MnADP complex and show that specific anions such as nitrate, formate, and thiocyanate decrease the apparent dissociation constant for creatine in its complex with the CH3S-blocked enzyme and MnADP, as is observed with native creatine kinase. However, the change in the PRR enhancement for the CH3S-enzyme-MnADP upon binding of creatine in the presence or absence of anions was appreciably smaller than for the native enzyme. For the H2NCOCH2-blocked enzyme, these anions failed to bring about any influence of creatine on the PRR enhancement. Consistent with the diminished influence of these anions on the PRR enhancement of the CH3S-enzyme-MnADP-creatine complex, EPR spectra of bound Mn(II) show that the CH3S-blocking group interferes with the pronounced anion-induced spectral changes which are observed with the native enzyme. EPR spectra for the H2NCOCH2-enzyme-MnADP complex were not influenced upon additions of creatine, even in the presence of anions. These results suggest that the altered catalytic properties of the CH3S-blocked enzyme arise from structural perturbations at the active site which are also reflected in the PRR enhancement factors and EPR spectral features of the Mn(II) complexes. Moreover, the results clearly indicate that the H2NCOCH2-blocking group, which completely inactivates the enzyme, also eliminates the ability of the MnADP site to sense the presence of the second substrate, creatine, alone and in combination with anions which are structural analogs of the migrating phosphoryl group.