Dehaye L, Alban C, Job C, Douce R, Job D
Unité Mixte CNRS/Rhône-Poulene, U. M. 41, Rhône-Poulenc Agrochimie, Lyon, France.
Eur J Biochem. 1994 Nov 1;225(3):1113-23. doi: 10.1111/j.1432-1033.1994.1113b.x.
Steady-state kinetics of the 220-kDa form of acetyl-CoA carboxylase (ACC220), as purified from mature pea seeds, have been investigated with respect to the substrate specificity and inhibition by quizalofop, a herbicide of the aryloxyphenoxypropionate type. The enzyme showed a dual specificity, being able to carboxylate propionyl-CoA at a maximal rate approximately 20% that measured in the presence of the acetyl-CoA substrate. These two reactions occur at separate sites on the enzyme. One site binds either acetyl-CoA (Km = 226 microM) or propionyl-CoA (Km = 38 microM) and is strongly inhibited by quizalofop (Ki = 25 microM and 9.3 microM for the acetyl-CoA and propionyl-CoA substrates, respectively). The other is specific for acetyl-CoA (Km = 11 microM) and is much less inhibited by quizalofop (Ki = 256 microM). Owing to the existence of these two catalytically different sites, the enzyme obeyed Michaelis-Menten kinetics with propionyl-CoA, but exhibited kinetic co-operativity in the presence of acetyl-CoA. Also, kinetics of propionyl-CoA carboxylase activity of ACC220 exhibited hyperbolic inhibition in the presence of quizalofop, but co-operative inhibition when following the ACC activity of the enzyme. The results suggest that the higher the substrate specificity, the lower the quizalofop sensitivity of the active site. Similar kinetic behaviour was observed with ACC220 purified from pea leaves. Also, the apparent correlation between the substrate specificity and the sensitivity of ACC towards quizalofop was confirmed by kinetic analyses of the low-molecular-mass form of ACC present in chloroplasts of young pea leaves. This enzyme was insensitive to quizalofop inhibition and was not able to carboxylate propionyl-CoA. No other propionyl-CoA carboxylase activity, different from that catalysed by ACC220, could be detected from either reproductive or vegetative organs of pea plants at any stage of development.
对从成熟豌豆种子中纯化得到的220 kDa形式的乙酰辅酶A羧化酶（ACC220）的稳态动力学进行了研究，涉及底物特异性以及精喹禾灵（一种芳氧苯氧丙酸酯类除草剂）对其的抑制作用。该酶表现出双重特异性，能够以约为乙酰辅酶A底物存在时最大速率20%的速率对丙酰辅酶A进行羧化。这两个反应发生在酶的不同位点。一个位点结合乙酰辅酶A（Km = 226 μM）或丙酰辅酶A（Km = 38 μM），并受到精喹禾灵的强烈抑制（乙酰辅酶A和丙酰辅酶A底物的Ki分别为25 μM和9.3 μM）。另一个位点对乙酰辅酶A具有特异性（Km = 11 μM），且受精喹禾灵的抑制作用小得多（Ki = 256 μM）。由于存在这两个催化特性不同的位点，该酶对丙酰辅酶A服从米氏动力学，但在乙酰辅酶A存在时表现出动力学协同性。此外，ACC220的丙酰辅酶A羧化酶活性动力学在精喹禾灵存在时表现出双曲线抑制，但在跟踪该酶的ACC活性时表现出协同抑制。结果表明，底物特异性越高，活性位点对精喹禾灵的敏感性越低。从豌豆叶片中纯化得到的ACC220也观察到了类似的动力学行为。此外，通过对幼嫩豌豆叶片叶绿体中存在的低分子量形式的ACC进行动力学分析，证实了底物特异性与ACC对精喹禾灵敏感性之间的明显相关性。这种酶对精喹禾灵抑制不敏感，并且不能对丙酰辅酶A进行羧化。在豌豆植株发育的任何阶段，从生殖器官或营养器官中均未检测到不同于ACC220催化的其他丙酰辅酶A羧化酶活性。
