La germination de la spore de clostridum tyrobutyricum: II. Démonstration de l'intervention de l'acétokinase et de la phosphotransacétylase par l'étude de leurs propriétés

Abstract

Summary The properties of an acetokinase AK (ATP: acetate phosphotransferase, E.C. 2.7.2.1) and a phosphotransacetylase PTA (acetyl-CoA: orthophosphate acetyl transferase, E.C. 2.3.1.8) isolated from vegetative cells of C. tyrobutyricum have been studied in order to investigate if they are involved in spore germination. These enzymes are similar to those from other microrganisms especially. E. coli for AK and C. kluyveri for PTA. AK has a molecular weight of 68,000 daltons. It phosphorylates acetate and at a smaller rate propionate. The K m values are 100 mM for acetate, 135 mM for propionate, 3 mM for ATP, 5 mM for ADP and 3 mM for acetyl-P. The equilibrium constant of the AK reaction, which is markedly in favor of ATP and acetate, has been estimated to be 0.005 and the mechanism has been found to be «Ping-Pong. AK has an absolute requirement for Mg 2+ or Mn 2+ ; it is strongly inhibited by Mg + and p-CMB and activated by reducing agents. The phosphorylation of acetate by AK is inhibited competitively by the following compounds: fluoroacetate > butyrate ≥ thioglycolate > lactate > glycolate and propionate; formate, glycine, iodoacetate and acetamide have no effect. PTA has a molecular weight of 58,000 daltons. The reaction catalyzed by PTA is reversible but the rate of acetylation of CoA is much greater than the hydrolysis of acetyl-CoA. The K m values are 1.1 mM for acetyl-P and 0.03 mM for CoA. K + and particularly NH 4 + activates PTA, but Na + , Li + , Cs + and Ca 2+ inhibit it. Na + is a competitive inhibitor with respect to NH 4 + . Enzyme activity is also dependent on the anions, the relative order of effectiveness is: Cl − > CH 3 -COO − > SO 4 2− > PO 4 3− . NADH, ADP, ATP and pyruvate display no significant effect on PTA but a non-competitive inhibition with acetyl-P was observed with 3 and 2.3-PGA and a mixed inhibition with 2-PGA. The comparison of cell and spores AK and PTA on the basis of their pH optima and their interaction with substrates and inhibitors did not show significant differences. It is concluded that AK and PTA are involved in the initiation of spore germination in C. tyrobutyricum because of the complete agreement between several properties of these enzymes, especially the effect of inhibitors, and those of germination. An hypothesis on the role of acetyl-CoA is suggested.