Kinetics of the Two Forms of Acetyl‐CoA Carboxylase from Pisum sativum

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Abstract
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.