Affinity labeling of creatine kinase by N-(2,3-epoxypropyl)-N-amidinoglycine.

The compound N-(2,3-epoxypropyl)-N-amidinoglytine (epoxycreatine) was synthesized to probe the active site of rabbit muscle creatine kinase (EC 2.7.3.2) because of its structural similarity to the normal substrate creatine and because of the reactivity of its epoxide moiety toward nucleophiles. In the presence of epoxycreatine and in the absence of other substrates, complete inhibition of enzymatic activity is observed which does not return after exhaustive dialysis. The inhibition follows saturation kinetics. At 0°C (pH 7.45), Iii”act is 335 mM compared to a KS value of 24 mM for creatine at 30°C. The inactivation half-time at saturating epoxycreatine concentration is 4.2 min. In a competitive manner, creatine protects the enzyme from inactivation by epoxycreatine. Product studies indicate that epoxycreatine is also a substrate for the enzymatic reaction in the presence of both M$+ and ATP. The partitioning of epoxycreatine between phosphorylation by MgATP and inactivation of the enzyme was followed spectrophotometrically at 25°C and found to be 15 turnovers/inactivating event. The inactivation half-time in the presence of MgATP is slightly greater than that when MgADP is present, which also is slightly greater than that of epoxycreatine alone. The so-called transition state analogue complex of MgADP2l NOsl