Abstract
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.
Highlights
“0 in adenosine 5’-[aj?-’’O,j?-”O&riphosphate in the E .MgADP, which in turn permits the formation of a further absence of creatine, in the presence of L-arginine or complex in which certain anions occupy the vacant site of the taurocyamine, or tirnansferable phosphoryl group between creatine and MgADP, the presenceof poor substrates wheresingle turnover experiments were performed.In order to support this prima facie evidence for an associative mechanism of phosphoryl transfer, an investigation was undertaken of l-carboxymethyl-2-aminoimidazolea,new substrate analogue of creatine
It is desirable to obtain stereochemical evidence imidazoline is a poor substrate for the phospho- to support thein-line mechanism, even if the above evidence rylation of MgADP by creatine kinase. These observa- is accepted there remains the possibility that phosphoryl tionscan beaccountedforbyan associative sN2(P) transfer within the ternary complex occurs by either an assomechanism of phosphoryl transfer and by a microen- ciative sN2(P) mechanism or by a dissociative sN1(P)mechvironment of the enzyme-bound creatine
To distinguish between these possibilities an experianalogue) which lowers tphKe, of the guanidino group ment is required which will indicate whetherthe P,OP, bond by several pH unitscomparedwiththat in aqueous of ATP is broken in the absence of creatine or in the presence solution
Summary
Substrate N-(aminoiminomethy1)-N-propylglycine,which has a Vmaxof approximately 1%of the value for creatine anda K,,, The equilibrium constant for the creatinekinase-catalyzed of approximately 50 mM [6]. The "P nmr spectrum of the dithiothreitol(100p ~ )to, protect thseulfhydryl groups of the ["03]ATP (notshown) isolated as before,showed that no enzyme[21]. Small anions, such as C1-, Nos-,HC03-, and. No evidence of '"0scrambling in the ["OO,]ATP wafsound in the "'P nmr spectrum of the isolated sample. An attractive substrate analogue for this purpose was l-carboxymethyl2-aminoimidazole (I) since its dihydro derivative, l-carboxymethyl-2-iminoimidazolidine[11], was known to be an excellent substrate for creatine kinase [13]. It was prepared as outlined in Scheme 2. NH:!.CH2.CH(OEt), ii, (a)NaOH, [6] HCl, (c)NH2.CN
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