Chemical modification of hydroxynitrile lyase by selective reaction of an essential cysteine‐SH group with α,β‐unsaturated propiophenones as pseudo‐substrates

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Abstract
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 inhibitor‐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‐phenylheptanoic acid, the reduced, linear addition product of the inhibitor to a cysteine‐SH group.