l-Propargylglycine, a naturally occurring γ,δ-acetylenic α-amino acid, induces mechanism-based inactivation of two pyridoxal phosphate dependent enzymes of methionine metabolism: (1) cystathionine γ-synthase, which catalyzes a γ-replacement reaction in methionine biosynthesis, and (2) methionine γ-lyase, which catalyzes a γ-elimination reaction in methionine breakdown. Biphasic pseudo-first-order inactivation kinetics were observed for both enzymes. Complete inactivation is achieved with a minimum molar ratio ([propargylglycine]/[enzyme monomer]) of 4:1 for cystathionine γ-synthase and of 8:1 for methionine γ-lyase, consistent with a small number of turnovers per inactivation event. Partitioning ratios were determined directly from observed primary kinetic isotope effects. [α-2H] Propargylglycine displays kH/kD values of about 3 on inactivation half-times. [α-3H]-Propargylglycine gives release of tritium to solvent nominally stoichiometric with inactivation but, on correction for the calculated tritium isotope discrimination, partition ratios of four and six turnovers per monomer inactivated are indicated for cystathionine γ-synthase and methionineγ-lyase, respectively. The inactivation stoichiometry, using [α-14C]-propargylglycine, is four labels per tetramer of cystathionine γ-synthase but usually only two labels per tetramer of methionine γ-lyase (half-of-the-sites reactivity). Two-dimensional urea isoelectrofocusing/NaDodSO4 electrophoresis suggests (1) that both native enzymes are α2β2 tetramers where the subunits are distinguishable by charge but not by size and (2) that, while each subunit of a cystathionine γ-synthase tetramer becomes modified by propargylglycine, only one a and one β subunit may be labeled in an inactive α2β2 tetramer of methionine γ-lyase. Steady-state spectroscopic analyses during inactivation indicated that modified cystathionine γ-synthase may reprotonate C2 of the enzyme-inactivator adduct, so that the cofactor is still in the pyridoxaldimine oxidation state. Fully inactivated methionine γ-lyase hasλmax values at 460 and 495 nm, which may represent conjugated pyridoximine paraquinoid that does not reprotonate at C2 of the bound adduct. Either species could arise from Michael-type addition of an enzymic nucleophile to an electrophilic 3,4-allenic paraquinoid intermediate, generated initially by propargylic rearrangement upon a 4,5-acetylenic pyridoximine structure, as originally proposed for propargylglycine inactivation of γ-cystathionase [Abeles, R., & Walsh, C. (1973) J. Am. Chem. Soc. 95, 6124], It is reasonable that cystathionine γ-synthase is the major in vivo target for this natural acetylenic toxin, the growth-inhibitory effects of which are reversed by methionine. © 1979, American Chemical Society. All rights reserved.
