The sulfonylurea herbicide chlorsulfuron and the imidazolinone herbicide imazaquin were shown to be noncompetitive and uncompetitive inhibitors, respectively, of purified acetolactate synthase from barley (Hordeum vulgare L.) with respect to pyrvuate. From double-reciprocal plots of the time-dependent biphasic inhibition by chlorsulfuron, an initial apparent inhibition constant of 68 nanomolar was calculated (a 0 to 4 minute hssay was used for the initial inhibition), and a final steady-state dissociation constant of 3 nanomolar was estimated. The corresponding constants for imazaquin were 10 and 0.55 micromolar. Specific binding of [14C]chlorsulfuron and [14C]imazaquin to purified acetolactate synthase from barley and partially purified enzyme from corn (Zea mays L.) could be demonstrated by gel filtration and equilibrium dialysis. Evidence is presented that the binding of the inhibitors to the enzyme follows the previously described mechanism of slow reversibility once excess inhibitor has been removed. However, after formation of the slowly reversible complex and subsequent dissociation, both chlorsulfuron and imazaquin seem to permanently inactivate acetolactate synthase. These results add a new feature to the mode of action of these herbicides with respect to their high herbicidal potency. ALS2 (EC 4.1.3.18; also referred to as acetohydroxyacid synthase) catalyzes the first common step in the plant biosynthetic pathway of the essential branched-chain amino acids. For valine and leucine biosynthesis, two molecules of pyruvate are condensed to form 2-acetolactate, while for isoleucine formation one molecule of pyruvate reacts with 2-ketobutyrate in a similar reaction. The enzyme has raised strong interest since structurally unrelated classes of modern and very potent herbicides and inhibitors, the sulfonylureas (16), the imidazolinones (1 8), the triazolo pyrimidines (20)

The adsorption protein g3p and another minor coat protein, g6p, are located at one end of the filamentous bacteriophage fd [Grant, R. A., Lin, T. C., Konigsberg, W. E. & Webster, R. E. (1981) J. Biol. Chem. 256,. Both proteins, representing the proximal tip, were detached as an entity by a technique that allowed for gentle solubilization. Disrupting the phage particle with the detergent sodium deoxycholate and chloroform dissociates the major coat protein g8p, frees the phage DNA, but leaves g3p and g6p associated with each other. The g3p-g6p complex, which we termed the adsorption complex, and an oligomeric form of g3p with lower molecular mass were isolated and purified by gel-filtration chromatography in the presence of deoxycholate. These different oligomeric structures of g3p showed a different mobility in non-denaturing polyacrylamidegel electrophoresis. Both forms were also found in non-denaturing polyacrylamide-gel electrophoresis from deoxycholate-and Triton-X-100-solubilized phage without prior chromatographic separation. The two oligomeric forms of g3p are composed of two g3p polypeptide chains in the case of the low-molecular-mass species, and four g3p and four g6p polypeptide chains for the adsorption complex.

In addition to the synthesis of ketolacids the enzyme acetolactate synthase shows an oxygen-consuming side reaction. Partially purified acetolactate synthase from corn (Zea mays L.) and barley (Hordeum vulgare L.) exhibits chemiluminescence in the presence of oxygen, Mn" and low concentrations of pyruvate. Light emission is inhibited by azide, but not by catalase or superoxide dismutase. The data suggest the formation of singlet oxygen during the catalytic cycle, and provides a basis for a highly sensitive assay for the oxygenase reaction of acetolactate synthase. Both synthase activity and chemiluminescence are inhibited by sulfonylurea herbicides. The results add a new aspect to the irreversible inhibition of acetolactate synthase by these herbicides which may be enhanced by the presence of reactive oxygen species.

The wild-type adsorption protein (g3p) of filamentous phage IKe cannot be exchanged with its analogous protein in the related Ff (M13, fd, and fl) phage particles. Deletion mutants of the protein, however, are assembled into Ff phage particles. These hybrid Ff phage particles bearing deleted IKe g3p attach to N pili, thus conserving the host attachment property of the protein but not its infection-initiating function. This means that the attachment specificity is determined by IKe g3p independently of other phage components in contact with it. Infection initiation function, the process in which phage DNA is released into the host, in contrast seems to require either more complex structural features of the protein (for example, a certain oligomeric structure) provided only in the original particle, or a concerted action of g3p with another particle component, not replaceable by its homologous counterpart in the related phage.

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