Proton liberation in the pre-steady state phase of creatine kinase.

Numerous stereochemical and kinetic investigations on the reaction pathway of creatine kinase (CK) suggest that this enzymic reaction proceeds via direct in-line transfer of phosphate between participating substrates and to date there has been no chemical evidence for any plausible intermediate between enzyme-substrate and enzyme-product complexes. By following the absorption pattern of a pH sensitive dye (o-cresol sulphonaphthalein) in a stopped flow module we have studied transient pH changes in the backward reaction of CK. While the rapid mixing of ADP and CK gives no pH transient, that of phosphocreatine (PCr) and CK gives H+ liberation with kapp of 62.8 sec-1. The magnitude of proton release is one H+ per monomer of CK. Mixing of PCr+CK with ADP does not give any detectable pH transient and the reaction immediately proceeds to steady phase. The mixing of ADP+CK with PCr again gives a release of 1.2 H+ per monomer of CK with kapp of around 67.2 sec-1 before the reaction proceeds to steady phase where there is absorption of one H+ per ADP transphosphorylated. The results obtained, therefore, indicate the involvement of proton deficient E.PCr and E.ADP.PCr complexes in the pathway of CK.

The denatured states of ribonuclease-A: mechanism of denaturation by lithium chloride.

Denaturation of ribonuclease-A by lithium chloride has been studied using difference spectral, circular dichroic and viscometric measurements. The difference spectral results were interpreted in the light of our observations that the solvent effect of the denaturant on the tyrosyl residue is non-linear. It has been observed that (1) the lithium chloride-denatured protein contains 3% alpha-helix and 18% beta-structure, and (2) only two of the three buried tyrosyl residues are normalized in the denatured protein.

Coupling of the enzymic activities of myosin ATPase and creatine kinase and its role in muscular contraction.

During muscular contraction the regeneration of ATP, catalysed by creatine kinase (CK), keeps pace with the hydrolysis of ATP by myosin ATPase posing the question of its regulatory mechanism. In the background of F-actin activation of heavy meromyosin (HMM) ATPase activity we have investigated in vitro the role of F-actin in regulating CK's activity in the absence and presence of HMM. For the coupled enzyme system we have also looked into the roles played by the individual reactants. F-actin has been found to appreciably increase CK's activity in the absence of HMM. While HMM alone inhibited CK's activity, there was a several fold increase when F-actin was also present. By a process of elimination we conclude that none of the reactants apart from H+ could be involved in regulating CK's activity in the coupled enzyme system. As no change in the pH of reaction mixture was observed during the reaction, we further conclude that the two enzymic reactions are coupled by proton transfer along F-actin. Implications of the findings for PCr-Cr shuttle and movements of ATP and ADP in sarcomere are discussed.