RESUMO
N-Hydroxy-N-isopropyloxamate (IpOHA) is known to inhibit extremely tightly (Ki of 22 pM) the bacterial acetohydroxy acid isomeroreductase (EC 1.1.1.86) [Aulabaugh and Schloss (1990) Biochemistry 29, 2824-2830], the second enzyme of the branched-chain-amino-acid-biosynthetic pathway. Yet, although the same pathway exists in plant cells, this compound presents only very poor herbicidal action. Towards the goal of gaining a better understanding of this behaviour, we have studied the mechanism of interaction of this compound with a highly purified acetohydroxy acid isomeroreductase of plant origin, i.e. the spinach (Spinacia oleracea) chloroplast enzyme. IpOHA behaved as a nearly irreversible inhibitor of the enzyme. Encounter complex formation was very slow (association rate constant 1.9 x 10(3) M-1.s-1) and involved a single bimolecular step. Since inhibition was competitive with respect to acetohydroxy acid substrates, the time needed to achieve substantial (90%) inhibition in vitro of enzyme activity in the simultaneous presence of substrates and inhibitors was extremely long (for example of the order of hours at 1 microM IpOHA and 100 microM acetohydroxy acid substrates). Thus, under in vivo conditions, binding of the inhibitor may be so slow that it may delay considerably the time required for inhibition of the target enzyme. Simialr kinetic behaviour was observed with another reaction intermediate analogue described by Schulz, Spönemann, Köcher and Wengenmayer [(1988) FEBS Lett. 238, 375-378], 2-dimethyl-phosphinoyl-2-hydroxyacetic acid (Hoe 704), which displays a higher herbicide activity than IpOHA. The herbicidal potency of these two compounds appeared to be correlated with their rates of association with the plant acetohydroxy acid isomeroreductase, since the bimolecular rate constant for Hoe 704 (2.2 x 10(4) M-1.s-1) was higher than that for IpOHA.