N‐Linoleyl amino acids as chiral probes for the substrate binding site of soybean lipoxygenase‐1

Lipoxygenases catalyze the oxygenation of polyunsaturated fatty acids and their derivatives to produce conjugated diene hydroperoxides. Soybean lipoxygenase‐1 (SBLO‐1) has been the subject of intensive structural and mechanistic study, but the manner in which this enzyme binds substrates is uncertain. In some models, the polar end of the substrate is deeply embedded in the protein; in others the polar end is on the surface (tail‐first binding). In this work we have studied the action of SBLO‐1 on N‐linoleoyl amino acids, which were found to be good substrates for the enzyme. Detailed studies have been carried out on N‐linoleoyl‐ l ‐valine (LLV) and N‐linoleoyl‐ d ‐valine (LDV). With each of these substrates, the predominant product is the 13( S )‐hydroperoxy‐ 9( Z ),11( E )‐diene, as in the case of linoleic acid, and the levels of regio‐ and stereospecificity are quite close to those observed with linoleic acid. Interestingly, the kinetic parameters for LLV (k cat = 205 ± 6 s −1 , K m = 8.9 ± 0.7 μM, k cat /K m = 23 ± 2 μM −1 s −1 ) are very close to those for LDV (k cat = 182 ± 7 s −1 , K m = 7.8 ± 0.7 μM, k cat /K m = 24 ± 3 μM −1 s −1 ) and comparable to the values for linoleic acid (k cat = 220 ± 13, K m = 13.3 ± 1.4 μM, k cat /K m = 16.6 ± 0.8 μM −1 s −1 ). The results indicate that the introduction of the valine moiety has little effect on substrate activity and that activity is insensitive to the stereochemical configuration of the valine. These findings suggest that the enzyme binds LLV and LDV in a manner that does not involve strong interaction between the valine moiety and the protein. The results are most easily accommodated by tail‐first binding models.