INTERACTIONS OF PLANT ACETOHYDROXY ACID ISOMEROREDUCTASE WITH REACTION INTERMEDIATE ANALOGS - CORRELATION OF THE SLOW, COMPETITIVE, INHIBITION-KINETICS OF ENZYME-ACTIVITY AND HERBICIDAL EFFECTS

N-Hydroxy-N-isopropyloxamate (IpOHA) is known to inhibit extremely tig htly (K-i 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 path way. Yet, although the same pathway exists in plant cells. this compou nd presents only very poor herbicidal action. Towards the goal of gain ing a better understanding of this behaviour, we have studied the mech anism of interaction of this compound with a highly purified acetohydr oxy acid isomeroreductase of plant origin, i.e. the spinach (Spinacia oleracea) chloroplast enzyme. IpOHA behaved as a nearly irreversible i nhibitor of the enzyme. Encounter complex formation was very slow (ass ociation rate constant 1.9 x 10(3) M(-1).s(-1)) and involved a single bimolecular step. Since inhibition was competitive with respect to ace tohydroxy 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 or der of hours at 1 mu M IpOHA and 100 mu M 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 o f the target enzyme. Similar kinetic behaviour was observed with anoth er reaction intermediate analogue described by Schulz, Sponemann, Koch er and Wengenmayer [(1988) FEBS Lett. 238, 375-378], 2-dimethyl-phosph inoyl-2-hydroxyacetic acid (Hoe 704), which displays a higher herbicid e activity than IpOHA. The herbicidal potency of these two compounds a ppeared to be correlated with their rates of association with the plan t acetohydroxy acid isomeroreductase, since the bimolecular rate const ant for Hoe 704 (2.2 x 10(-4) M(-1).s(-1)) was higher than that for Ip OHA.