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.