KINETIC-STUDIES ON 2 ISOFORMS OF ACETYL-COA CARBOXYLASE FROM MAIZE LEAVES

The steady-state kinetics of two multifunctional isoforms of acetyl-Co A carboxylase (ACCase) from maize leaves (a major isoform, ACCase1 and a minor isoform, ACCase2) have been investigated with respect to reac tion mechanism, inhibition by two graminicides of the aryloxyphenoxypr opionate class (quizalofop and fluazifop) and some cellular metabolite s. Substrate interaction and product inhibition patterns indicated tha t ADP and P-i products from the first partial reaction were not releas ed before acetyl-CoA bound to the enzymes. Product inhibition patterns did not match exactly those predicted for an ordered Ter Ter or a ran dom Ter Ter mechanism, but were close to those postulated for an order ed mechanism. ACCase2 was about 1/2000 as sensitive as ACCase1 to quiz alofop but only about 1/150 as sensitive to fluazifop. Fitting inhibit ion data to the Hill equation indicated that binding of quizalofop or fluazifop to ACCase1 was non-cooperative, as shown by the Hill constan t (n(app)) values of 0.86 and 1.16 for quizalofop and fluazifop respec tively. Apparent inhibition constant values (K' from the Hill equation ) for ACCase1 were 0.054 mu M for quizalofop and 21.8 mu M for fluazif op. On the other hand, binding of quizalofop or fluazifop to ACCase2 e xhibited positive co-operativity, as shown by the n(app) values of 1.8 5 and 1.59 for quizalofop and fluazifop respectively. K' values for AC Case2 were 1.7 mM for quizalofop and 140 mM for fluazifop. Kinetic par ameters for the co-operative binding of quizalofop to maize ACCase2 we re close to those of another multifunctional ACCase of limited sensiti vity to graminicide, ACC220 from pea; Inhibition of ACCase1 by quizalo fop was mixed-type with respect to acetyl-CoA or ATP, but the concentr ation of acetyl-CoA had the greater effect on the level of inhibition. Neither ACCase1 nor ACCase2 was appreciably sensitive to CoA esters o f palmitic acid (16:0) or oleic acid (18:1). Approximate IC50 values w ere 10 mu M (ACCase2) and 50 mu M (ACCase1) for both CoA esters. Citra te concentrations up to 1 mM had no effect on ACCase1 activity. Above this concentration, citrate was inhibitory. ACCase2 activity was sligh tly stimulated by citrate over a broad concentration range (0.25-10 mM ). The significance of possible effects of acyl-CoAs or citrate in viv o is discussed.