TRANSIENT INACTIVATION OF ALMOND MANDELONITRILE LYASE BY 3-METHYLENEOXINDOLE - A PHOTOOXIDATION PRODUCT OF THE NATURAL PLANT HORMONE INDOLE-3-ACETIC-ACID
Ip. Petrounia et al., TRANSIENT INACTIVATION OF ALMOND MANDELONITRILE LYASE BY 3-METHYLENEOXINDOLE - A PHOTOOXIDATION PRODUCT OF THE NATURAL PLANT HORMONE INDOLE-3-ACETIC-ACID, Biochemistry, 33(10), 1994, pp. 2891-2899
A variety of plant growth regulators belonging to the auxin phytohormo
ne family have been found to be good competitive inhibitors of the oxy
nitrilase from almonds, mandelonitrile lyase (MNL). The major natural
auxin, indole-3-acetic acid (IAA), was found to inactivate MNL in a re
action following pseudo-first-order kinetics and dependent upon visibl
e light. Inactivation results from the oxidative decarboxylation of IA
A forming 3-methyleneoxindole (MOI). This compound has been synthesize
d 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 condit
ions, in reasonable agreement with the value obtained from the inactiv
ation data. Several indole derivatives such as indoline, skatole, oxin
dole, and 3-methyloxindole are poor competitive inhibitors of MNL with
dissociation constants 20-40-fold greater than that for MOI, suggesti
ng 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 quantit
ative recovery occurs in the presence of 2-mercaptoethanol or DTT, ind
icating that the adduct is labile. The effect of pH on the inactivatio
n reaction suggests modification of a single amino acid functional gro
up with an apparent pK(a) of 5.6 +/- 0.03. The inactivation data may b
e explained by a mechanism involving 1,4-conjugate addition of a prote
in functional group to form an unstable Michael adduct, which is capab
le of dissociating from the enzyme following dilution or after consump
tion of excess MOI by added thiols.