Transient Inactivation of Almond Mandelonitrile Lyase by 3-Methyleneoxindole: A Photooxidation Product of the Natural Plant Hormone Indole-3-Acetic Acid

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Abstract
A variety of plant growth regulators belonging to the auxin phytohormone family have been found to be good competitive inhibitors of the oxynitrilase from almonds, mandelonitrile lyase (MNL). The major natural auxin, indole-3-acetic acid (IAA), was found to inactivate MNL in a reaction following pseudo-first-order kinetics and dependent upon visible light. Inactivation results from the oxidative decarboxylation of IAA forming 3-methyleneoxindole (MOI). This compound has been synthesized and shown to produce active-site-directed inactivation of MNL, in a reaction following saturation kinetics with a K-I of 37 +/- 8 mu M and maximal k(inact) of 0.13 +/- 0.02 min(-1). Inactivation protection is provided by the competitive inhibitors azide and benzoate, suggesting that the inactivation reaction is active-site-directed. This idea is substantiated by our determination that MOI is a competitive inhibitor of MNL with a K-i of 23 +/- 3 mu M under steady-state turnover conditions, in reasonable agreement with the value obtained from the inactivation data. Several indole derivatives such as indoline, skatole, oxindole, and 3-methyloxindole are poor competitive inhibitors of MNL with dissociation constants 20-40-fold greater than that for MOI, suggesting a highly specific binding site for the IAA photooxidation product. The enzyme remains inactive following spin dialysis, indicating that a covalent adduct has been formed. However, approximately 30% activity was recovered in a 5-h period following dialysis, and a nearly quantitative recovery occurs in the presence of 2-mercaptoethanol or DTT, indicating that the adduct is labile. The effect of pH on the inactivation reaction suggests modification of a single amino acid functional group with an apparent pK(a) of 5.6 +/- 0.03. The inactivation data may be explained by a mechanism involving 1,4-conjugate addition of a protein functional group to form an unstable Michael adduct, which is capable of dissociating from the enzyme following dilution or after consumption of excess MOI by added thiols.