Suicide inactivation of bacterial cystathionine .gamma.-synthase and methionine .gamma.-lyase during processing of L-propargylglycine

Abstract

L-Propargylglycine, a naturally occurring .gamma.,.delta.-acetylenic .alpha.-amino acid, induces mechanism-based inactivation of 2 pyridoxal phosphate dependent enzymes of methionine metabolism: cystathionine .gamma.-synthase, which catalyzes a .gamma.-replacement reaction in methionine biosynthesis, and methionine .gamma.-lyase, which catalyzes a .gamma.-elimination reaction in methionine breakdown. Biphasic pseudo-1st-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 .gamma.-synthase and of 8:1 for methionine .gamma.-lyase, consistent with a small number of turnovers per inactivation event. Partitioning ratios were determined directly from observed primary kinetic isotope effects. [.alpha.-2H]Propargylglycine displays kH/kD values of about 3 on inactivation half-times. [.alpha.-3H]-Propargylglycine gives release of tritium to solvent nominally stoichiometric with inactivation but, on correction for the calculated tritium isotope discrimination, partition ratios of 4 and 6 turnovers/monomer inactivated are indicated for cystathionine .gamma.-synthase and methionine .gamma.-lyase, respectively. The inactivation stoichiometry, using [.alpha.-14C]-propargylglycine, is 4 labels/tetramer of cystathionine .gamma.-synthase but usually only 2 labels/tetramer of methionine .gamma.-lyase (half-of-the-sites reactivity). Two-dimensional urea isoelectrofocusing/sodium dodecyl sulfate electrophoresis suggests that both native enzymes are .alpha.2.beta.2 tetramers where the subunits are distinguishable by charge but not by size and that, while each subunit of a cystathionine .gamma.-synthase tetramer becomes modified by proparglyglycine, only one .alpha. and one .beta. subunit may be labeled in an inactive .alpha.2.beta.2 tetramer of methionine .gamma.-lyase. Steady-state spectroscopic analyses during inactivation indicated that modified cystathionine .gamma.-synthase may reprotonate C2 of the enzyme-inactivator adduct, so that the cofactor is still in the pyridoxaldimine oxidation state. Fully inactivated methionine .gamma.-lyase has .lambda.max 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 proparglyglycine inactivation of .gamma.-cystathionase. Cystathionine .gamma.-synthase is the major in vivo target for this natural acetylenic toxin, the growth-inhibitory effects of which are reversed by methionine. [Salmonella typhimurium was used.].