Chemical Modification of Hydroxynitrile Lyase by Selective Reaction of an Essential Cysteine-SH Group with Alpha, Beta-unsaturated Propiophenones As Pseudo-substrates

3-Oxo-3-phenylpropyne and 3-oxo-3-phenylpropene were synthesized as active-site-directed irreversible inhibitors of the bitter almond hydroxynitrile lyase (EC 4.1.2.10), an FAD-protein. The substrate and competitors (e.g. benzoate) decrease the rate of the inhibitor-mediated deactivation of the enzyme. By excess addition of either one of the two inhibitors, the deactivation process is shown to be pseudo-first order. The reaction with equimolar amounts of 3-oxo-3-phenylpropyne with the enzyme is accompanied by a shift in the ultraviolet spectrum of the inhibitor, allowing direct measurement of the enzyme-inactivation process. The spectral change has second-order kinetics. Incubation with 3-oxo-3-[p-3H]phenylpropyne or 3-oxo-3-[1-14C]phenylpropene shows a one-to-one stoichiometry for the inhibitory-enzyme reaction. Dissociation of the 3-oxo-3[p-3H]phenylpropyne-inactivated holoenzyme with acid ammonium sulfate yields a labeled apoenzyme; the inhibitor does not react with free or enzyme-bound FAD. After boranate reduction and exhaustive hydrolysis of the 3-oxo-3-[1-14C]phenylpropene-inactivated enzyme, a labeled cysteine derivative was isolated which was identified by chromatographic and mass spectroscopic comparison with synthetic references as L-2-amino-4-thia-DL-7-hydroxy-7-phenylhepatanoic acid, the reduced, linear addition product of the inhibitor to a cysteine-SH group.