Evidence about the Role of the Essential Thiol Groups in Creatine Kinase from Differential Thermal Analysis

Abstract

Creatine kinase (ATP-creatine phosphotransferase, EC 2.7.3.2) contains two reactive thiol groups/molecule (Benesch, Lardy & Benesch,1955) that may be specifically alkylated with concomitant loss of enzyme activity by iodoacetate (Watts, Rabin & Crook, 1961) or by iodoacetamide (D. C. Watts, unpublished work). On the basis of these and other data Watts & Rabin (1962) suggested that each thiol group may be hydrogen-bonded to a histidine residue and plays an important role in the catalytic reaction by interacting with the substrates, ATP-Mg2+ and creatine. Additional evidence based on the inactivation of the enzyme by Mn2+ supports this hypothesis (Watts, 1963). During the catalytic reaction conformational changes in the enzyme are also thought to occur (Samuels, Nihei & Noda, 1961; Watts, 1963) and the possibility exists that the thiol groups are also implicated in this process, perhaps rather than in the catalytic mechanism. The present communica- tion reports thermal-denaturation experiments that partly help to resolve this problem.