A role for glutamate-333 of Saccharomyces cerevisiae cystathionine γ-lyase as a determinant of specificity

Abstract

Cystathionine γ-lyase (CGL) catalyzes the hydrolysis of l-cystathionine (l-Cth), producing l-cysteine (l-Cys), α-ketobutyrate and ammonia, in the second step of the reverse transsulfuration pathway, which converts l-homocysteine (l-Hcys) to l-Cys. Site-directed variants substituting residues E48 and E333 with alanine, aspartate and glutamine were characterized to probe the roles of these acidic residues, conserved in fungal and mammalian CGL sequences, in the active-site of CGL from Saccharomyces cerevisiae (yCGL). The pH optimum of variants containing the alanine or glutamine substitutions of E333 is increased by 0.4-1.2 pH units, likely due to repositioning of the cofactor and modification of the pKa of the pyridinium nitrogen. The pH profile of yCGL-E48A/E333A resembles that of Escherichia coli cystathionine β-lyase. The effect of substituting E48, E333 or both residues is the 1.3-3, 26-58 and 124-568-fold reduction, respectively, of the catalytic efficiency of l-Cth hydrolysis. The Km(l-Cth) of E333 substitution variants is increased ~17-fold, while Km(l-OAS) is within 2.5-fold of the wild-type enzyme, indicating that residue E333 interacts with the distal amine moiety of l-Cth, which is not present in the alternative substrate O-acetyl-l-serine. The catalytic efficiency of yCGL for α,γ-elimination of O-succinyl-l-homoserine (kcat/Km(l-OSHS)=7±2), which possesses a distal carboxylate, but lacks an amino group, is 300-fold lower than that of the physiological l-Cth substrate (kcat/Km(l-Cth)=2100±100) and 260-fold higher than that of l-Hcys (kcat/Km(l-Hcys)=0.027±0.005), which lacks both distal polar moieties. The results of this study suggest that the glutamate residue at position 333 is a determinant of specificity.

Keywords: 1-aminocyclopropane-1-carboxylate synthase; 2-(Bis(2-hydroxyethyl)amino)acetic acid; 3-(Cyclohexylamino)-1-propanesulfonic acid; 5,5′-Dithiobis(2-nitrobenzoic acid); AAT; ACCS; AMPSO; CAPS; CBL; CGL; CGS; DTNB; HO-HxoDH; IPTG; LDH; MGL; N-(1,1-Dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid; N-[Tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid; NADH (reduced form); Ni-NTA; Ni-nitrilo triacetic acid; O-acetyl-l-serine; O-succinyl-l-homoserine; OAS sulfhydrylase; OASS; PLP; Pyridoxal 5'-phosphate; Reaction specificity; Structure–function relationships; TAPS; aspartate aminotransferase; bicine; cystathionine β-lyase; cystathionine γ-lyase; cystathionine γ-synthase; d-2-hydroxyisocaproate dehydrogenase; isopropyl-β-D-thiogalactopyranoside; l-Cth; l-Hcys; l-OAS; l-OSHS; l-cystathionine; l-homocysteine; l-lactate dehydrogenase; methionine γ-lyase; pyridoxal 5′-phosphate; β-nicotinamide adenine dinucleotide.