J. Biol. Chem., Vol. 255, Issue 9, 3944-3951, May, 1980
Evidence for an associative mechanism in the phosphoryl transfer step catalyzed by rabbit muscle creatine kinase
G Lowe and BS Sproat
Creatine kinase does not catalyze the scrambling of 18O in adenosine 5'-
[alpha beta-18O, beta-18O2]triphosphate in the absence of creatine, in the
presence of L-arginine or taurocyamine (competitive inhibitors of
creatine), or in the presence of poor substrates where single turnover
experiments were performed. In order to support this prima facie evidence
for an associative mechanism of phosphoryl transfer, an investigation was
undertaken of 1-carboxymethyl-2-aminoimidazole, a new substrate analogue of
creatine. This analogue has a binding constant for rabbit muscle creatine
kinase similar to creatine and 1- carboxymethyl-2-iminoimidazolidine, but
the initial rate of phoshorylation by MgATP in the presence of creatine
kinase is almost 5 orders of magnitude slower. The phosphorylation product,
assigned the structure 1-carboxymethyl-2-imino-3-phospho-4-imidazoline is
also a poor substrate for the phosphorylation of MgADP by creatine kinase.
These observations can be accounted for by an associative SN2(P) mechanism
of phosphoryl transfer and by a microenvironment of the enzyme-bound
creatine (or creatine analogue) which lowers the pKa of the guanidino group
by several pH units compared with that in aqueous solution.
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