Interactions of plant acetohydroxy acid isomeroreductase with reaction intermediate analogues: correlation of the slow, competitive, inhibition kinetics of enzyme activity and herbicidal effects.

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.

Effect of glyphosate on plant cell metabolism. 31P and 13C NMR studies.

The effect of glyphosate (N-phosphonomethyl glycine; the active ingredient of Roundup herbicide) on plant cells metabolism was analysed by 31P and 13C NMR using suspension-cultured sycamore (Acer pseudoplatanus L) cells. Cells were compressed in the NMR tube and perfused with an original arrangement enabling a tight control of the circulating nutrient medium. Addition of 1 mM glyphosate to the nutrient medium triggered the accumulation of shikimate (20-30 mumol g-1 cell wet weight within 50 h) and shikimate 3-phosphate (1-1.5 mumol g-1 cell wet weight within 50 h). From in vivo spectra it was demonstrated that these two compounds were accumulated in the cytoplasm where their concentrations reached potentially lethal levels. On the other hand, glyphosate present in the cytoplasmic compartment was extensively metabolized to yield aminomethylphosphonic acid which also accumulated in the cytoplasm. Finally, the results presented in this paper indicate that although the cell growth was stopped by glyphosate the cell respiration rates and the level of energy metabolism intermediates remained unchanged.

Transient accumulation of asparagine in sycamore cells after a long period of sucrose starvation.

The mobilization of stored carbohydrates (sucrose and starch) and proteins during sucrose starvation was studied with sycamore (Acer pseudoplatanus L.) cells. When almost all the intracellular carbohydrate pools had disappeared, the cell protein content declined progressively whereas asparagine determined by either (13)C nuclear magnetic resonance or reversed phase high performance liquid chromatography increased steadily. After a long period of sucrose starvation, the most intense resonances in the (13)C nuclear magnetic resonance spectra were from citrate and asparagine. The total amounts of asparagine (expressed as nitrogen) and free amino acids that appeared after a long period of sucrose deprivation corresponded roughly to the total amount of protein (expressed as nitrogen), that disappeared within the same period of time. Addition of sucrose in the culture medium after a long period of sucrose starvation led to a disappearance of asparagine. These results suggest therefore that the presence of asparagine in plant cells in large excess should be considered as a good marker of protein utilization after a long period of sucrose starvation and is very likely related to stress.