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 two pyridoxal phosphate dependent enzymes of methionine metabolism: (1) cystathionine gamma-synthease, which catalyzes a gamma-replacement reaction in methionine biosynthesis, and (2) methionine gamma-lyase, which catalyzes a gamma-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 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 four and six turnovers per monomer inactivated are indicated for cystathionine gamma-synthase and methionine gamma-lyase, respectively. The inactivation stoichiometry, using [alpha-14C]-propargylglycine, is four labels per tetramer of cystathionine gamma-synthase but usually only two labels per tetramer of methionine gamma-lyase (half-of-the-sites reactivity). Two-dimensional urea isoelectrofocusing/NaDodSO4 electrophoresis suggests (1) that both native enzymes are alpha 2 beta 2 tetramers where the subunits are distinguishable by charge but not by size and (2) that, while each subunit of a cystathionine gamma-synthase tetramer becomes modified by propargylglycine, 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 propargylglycine inactivation of gamma-cystathionase [Abeles, R., & Walsh, C. (1973) J. Am. Chem. Soc. 95, 6124]. It is reasonable that cystathionine gamma-synthase is the major in vivo target for this natural acetylenic toxin, the growth-inhibitory effects of which are reversed by methionine.

Functional Demonstration of Reverse Transsulfuration in the Mycobacterium tuberculosis Complex Reveals That Methionine Is the Preferred Sulfur Source for Pathogenic Mycobacteria

“…The dithioerythritol was not transformed in any of the assays conducted (results not shown). Reactions were started by the addition of cell-free extract except when the effect of propargylglycine was tested (26,27). Then, extracts were preincubated with propargylglycine (range of concentrations: 4 -100 M) for 30 min at 37°C before starting the reaction by adding substrate.…”

Identification and Characterization of Two Isoenzymes of Methionine γ-Lyase from Entamoeba histolytica

“…Inhibition of MGL by PPG-To better understand the biochemical characteristics of EhMGL, we evaluated effects of DL-propargylglycine (PPG), a potent inhibitor of the ␥-subfamily of PLP-dependent enzymes (46,47), on the recombinant EhMGLs. Activity of both rEhMGL1 and 2 was inhibited by PPG in an irreversible and slow-binding manner (Fig.…”

PLP-Dependent Enzymes: a Potent Therapeutic Approach for Cancer and Cardiovascular Diseases

“…LMethioninase displays two absorption maxima at 280 nm, as aromatic amino acids containing protein, and at 420 nm due to the internal aldimine linkage of the aldhyde group of pyridoxal phosphate and Ɛ-amino group of lysine N-terminal domain of PLP-enzyme. The absorption spectra of bacterial and fungal L-methioninase (Tanaka et al, 1977, Johnston et al, 1979, El-Sayed, 2011) are typically to other pyridoxal-dependent enzymes (Bertoldi et al, 2002;Saha et al, 2009). The pyridoxal phosphate co-enzyme was dissociate from the apoenzyme by incubation with 10 mM hydroxylamine, giving one peak at 280nm, with disappearance of the 420 nm peak due to the dissociation of the PLP and hydrolysis of the internal aldimine bond.…”

“…The ratio of A280/420 was decreased from 4.01 to 1.7, upon addition of pyridoxal phosphate (0.2 mM) to the apo-enzyme, indicating the full reconstitution of the active holo-enzyme (El-Sayed, 2011) (Fig.5). Johnston et al (1979) reported that upon freezing and thawing the ratio of A280/420 of Pseudomonas ovalis Lmethioninase was increased from 3.90 to 4.7, due to the dissociation of pyridoxal phosphate. P. ovalis L-methioninase lacks the ability to restore its original activity by dialysis against pyridoxal phosphate (Johnston et al, 1979), while that of Trichomonas vaginalis enzyme restore more than 90 % of its activity by 0.1mM PLP (Lockwood and Coombs, 1991).…”

“…Johnston et al (1979) reported that upon freezing and thawing the ratio of A280/420 of Pseudomonas ovalis Lmethioninase was increased from 3.90 to 4.7, due to the dissociation of pyridoxal phosphate. P. ovalis L-methioninase lacks the ability to restore its original activity by dialysis against pyridoxal phosphate (Johnston et al, 1979), while that of Trichomonas vaginalis enzyme restore more than 90 % of its activity by 0.1mM PLP (Lockwood and Coombs, 1991). However, A. flavipes L-methioninase has the ability to reconstitute its fully structural catalytic state upon addition of pyridoxal phosphate (0.2mM), similarly to cystathioninelyase (Zhu et al, 2008).…”