Comparaison des groupes SH reactifs des ATP:guanidines phosphotransferases

Abstract

Comparative study of the reactive SH groups of ATP: guanidine phosphotransferases 1. 1. With 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) in the presence of urea, 6 SH groups are titrated in ATP:creatine phosphotransferase (EC 2.7.3.2), ATP:arginine phosphotransferase (EC 2.7.3.3) and ATP:lombricine phosphotransferase (EC 2.7.3.5); 14 SH groups are found in ATP:taurocyamine phosphotransferase (EC 2.7.3.4) and 16 in ATP:guanidinoacetate phosphotransferase (EC 2.7.3.1). In the absence of urea, only 1 SH group is titrated in lombricine kinase, 2 in creatine and taurocyamine kinases, and 6 in arginine and glycocyamine kinases. 2. 2. At pH 8.5, 3 of the SH groups of arginine kinase react at once with DTNB; at pH 7, only one SH is immediately titratable. The blocking of this SH group results in the complete inhibition of the enzyme. l-Arginine efficiently antagonizes the inhibitor; Mg-ATP and Mg-ADP decrease the rate of alkylation but do not restore the inactivation of arginine kinase. 3. 3. Stoichiometric inhibition by DTNB reveals the presence of one reactive SH group in arginine and lombricine kinases and of 2 essential SH groups in glycocyamine, creatine and taurocyamine kinases. Guanidine substrates, arginine and lombricine, efficiently protect their respective enzymes against inactivation by DTNB; in contrast glycocyamine and creatine do not protect their corresponding enzymes. Moreover, Mg-ATP protects glycocyamine and creatine kinases but not the arginine and lombricine kinases. Taurocyamine kinase is intermediate between these 2 groups of enzymes: it is protected from DTNB by both guanidine and nucleotide substrates. 4. 4. Fingerprints of N- [I- 14C]ethylmaleimide-labelled creatine, taurocyamine and lombricine kinases show the presence of approximately the same number of peptides, 46, which are mainly neutral or basic. Arginine kinase, the molecular weight of which is half that of the other 3 enzymes, produces nearly the same number of peptides, 45–48. On autoradiograms of the fingerprints of the 4 enzymes, the main radioactivity seems to be situated in the same peptide.