Mechanism of inhibition of sterol biosynthesis enzymes by N‐substituted morpholines

Tridemorph, fenpropimorph and derivatives have been tested in vitro on Δ 8 →Δ 7 ‐sterol isomerase (SI) from maize seedlings. The results show that these N ‐substituted morpholines strongly inhibit this enzyme (I 50 : 0.4 μM for fenpropimorph, 0.6 μM for tridemorph). In fenpropimorph, where a chiral centre is present at C‐2 of the alkyl chain, good enantioselectivity was observed for enzyme inhibition. Inhibition of SI and of the cycloeucalenol‐obtusifoliol isomerase (COI), another enzyme of plant sterol biosynthesis, is probably due to protonation of the N ‐substituted morpholines, giving a positively charged nitrogen atom, at the pH (7.4) of the enzyme assays. In order to test this hypothesis, the pH dependence of the inhibition constants has been measured. Fenpropimorph, (pK a 7.5) which can be protonated to give a morpholinium cation and N ‐methylfenpropimorph, the corresponding quaternary ammonium derivative, have been tested as inhibitors in a pH range from 6 to 8.5. The I 50 value of fenpropimorph toward the SI increased 20 times as the pH increased from 6 to 8.5. In contrast the I 50 value of fenpropimorph for COI did not change significantly in this pH range. While the I 50 value of N ‐methyl fenpropimorph towards COI decreased more than 50 times as the pH increased from 6 to 8.5, its I 50 value for SI only varied slightly in this pH range. Taken together, these results suggest the existence of an interaction of the morpholinium cations with one or several enzyme amino acid residues of a much higher pK a in the case of COI than SI. Moreover for maize COI, the pK a of the amino acid residue(s) interacting with these morpholines derivatives is probably close to that of these molecules.