PROTON LIBERATION IN THE PRE-STEADY-STATE PHASE OF CREATINE-KINASE

Numerous stereochemical and kinetic investigations on the reaction pat hway of creatine kinase (CK) suggest that this enzymic reaction procee ds via direct in-line transfer of phosphate between participating subs trates and to date there has been no chemical evidence for any plausib le intermediate between enzyme-substrate and enzyme-product complexes. By following the absorption pattern of a pH sensitive dye (o-cresol s ulphonaphthalein) in a stopped flow module we have studied transient p H changes in the backward reaction of CK. While the rapid mixing of AD P and CK gives no pH transient, that of phosphocreatine (PCr) and CK g ives H+ liberation with k(app) 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 proc eeds to steady phase. The mixing of ADP+CK with PCr again gives a rele ase of 1.2 H+ per monomer of CK with k(app) of around 67.2 sec-1 befor e the reaction proceeds to steady phase where there is absorption of o ne H per A DP transphosphorylated. The results obtained, therefore, in dicate the involvement of proton deficient E.PCr and F.ADP.PCr complex es in the pathway of CK.