Cystathionine gamma-synthase (CGS) and cystathionine beta-lyase (CBL), which comprise the transsulfuration pathway of bacteria and plants, and cystathionine gamma-lyase (CGL), the second enzyme of the fungal and animal reverse transsulfuration pathway, share approximately 30% sequence identity and are almost indistinguishable in overall structure. One difference between the active site of Escherichia coli CBL and those of E. coli CGS and Saccharomyces cerevisiae CGL is the replacement of a pair of aromatic residues, F55 and Y338, of the former by acidic residues in CGS (D45 and E325) and CGL (E48 and E333). A series of interconverting, site-directed mutants of these 2 residues was constructed in CBL (F55D, Y338E, F55D/Y338E), CGS (D45F, E325Y and D45F/E325Y) and CGL (E48A,D and E333A,D,Y) to probe the role of these residues as determinants of reaction specificity. Mutation of either position results in a reduction in catalytic efficiency, as exemplified by the 160-fold reduction in the kcat/KmL-Cys of eCGS-D45F and the 2850- and 30-fold reductions in the kcat/KmL-Cth of the eCBL-Y338E and the yCGL-E333A,Y mutants, respectively. However, the in vivo reaction specificity of the mutants was not altered, compared with the corresponding wild-type enzymes. The DeltametB and DeltametC strains, the optimized CBL and CGL assay conditions, and the efficient expression and affinity purification systems described provide the necessary tools to enable the continued exploration of the determinants of reaction specificity in the enzymes of the transsulfuration pathways.

Cystathionine β-lyase (CBL) catalyzes the hydrolysis of L-cystathionine (L-Cth) to produce L-homocysteine, pyruvate, and ammonia. A series of site-directed variants of Escherichia coli CBL (eCBL) was constructed to investigate the roles of the hydroxyl moieties of active-site residues Y56, Y111, Y238, Y338, and S339 as determinants of specificity. The effect of these conservative substitutions on the k(cat)/K(m)(L-Cth) for the α,β-elimination of L-Cth ranges from a change of only 1.1-fold for Y338F to a reduction of 3 orders of magnitude for the alanine replacement variant of S339. A novel role for residue S339 as a determinant of reaction specificity, via tethering of the catalytic base, K210, is demonstrated. Comparison of the kinetic parameters for L-Cth hydrolysis with those for the inhibition of eCBL by aminoethoxyvinylglycine (AVG) indicates that Y238 interacts with the distal carboxylate group of the substrate. The 22 and 50-fold increases in the K(m)(L-Cth) and K(i)(AVG) resulting from replacement of Y56 with phenylalanine suggest that this residue may interact with the distal amino group of these compounds, although an indirect role in binding is more likely. The near-native k(cat)/K(m)(L-Cth) and pH profile of the eCBL-Y111F variant demonstrate that residue Y111 does not play a role in proton transfer. The understanding of the eCBL active site and of the determinants of substrate and reaction specificity resulting from this work will facilitate the design of inhibitors, as antibacterial therapeutics, and the engineering of enzymes dependent on the catalytically versatile pyridoxal 5'-phosphate cofactor to modify reaction specificity.

Cystathionine b-lyase (CBL) catalyzes the hydrolysis of L-cystathionine (L-Cth) to produce L-homocysteine, pyruvate, and ammonia. A series of active-site mutants of Escherichia coli CBL (eCBL) was constructed to investigate the roles of residues R58, R59, D116, W340, and R372 in catalysis and inhibition by aminoethoxyvinylglycine (AVG). The effects of these mutations on the k cat /K LÀCth m for the b-elimination reaction range from a reduction of only 3-fold for D116A and D116N to 6 orders of magnitude for the R372L and R372A mutants. The order of importance of these residues for the hydrolysis of L-Cth is: R372 >> R58 > W340 % R59 > D116. Comparison of the kinetic parameters for L-Cth hydrolysis with those for inhibition of eCBL by AVG demonstrates that residue R58 tethers the distal carboxylate group of the substrate and confirms that residues W340 and R372 interact with the a-carboxylate moiety. The increase in the pK a of the acidic limb and decrease in the pK a of the basic limb of the k cat /K LÀCth m versus pH profiles of the R58K and R58A mutants, respectively, support a role for this residue in modulating the pK a of an active-site residue.

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Cystathionine c-synthase (CGS) catalyzes the condensation of O-succinyl-L-homoserine (L-OSHS) and L-cysteine (L-Cys), to produce L-cystathionine (L-Cth) and succinate, in the first step of the bacterial transsulfuration pathway. In the absence of L-Cys, the enzyme catalyzes the futile a,c-elimination of L-OSHS, yielding succinate, a-ketobutyrate, and ammonia. A series of 16 sitedirected variants of Escherichia coli CGS (eCGS) was constructed to probe the roles of active-site residues D45, Y46, R48, R49, Y101, R106, N227, E325, S326, and R361. The effects of these substitutions on the catalytic efficiency of the a,c-elimination reaction range from a reduction of only~2-fold for R49K and the E325A,Q variants to 310-and 760-fold for R361K and R48K, respectively. A similar trend is observed for the k cat /K l-OSHS m of the physiological, a,c-replacement reaction. The results of this study suggest that the arginine residues at positions 48, 106 and 361 of eCGS, conserved in bacterial CGS sequences, tether the distal and a-carboxylate moieties, respectively, of the L-OSHS substrate. In contrast, with the exception of the 13-fold increase observed for R106A, the K l-Cys m is not markedly affected by the site-directed replacement of the residues investigated. The decrease in k cat observed for the S326A variant reflects the role of this residue in tethering the side chain of K198, the catalytic base. Although no structures exist of eCGS bound to active-site ligands, the roles of individual residues is consistent with the structures inhibitor complexes of related enzymes. Substitution of D45, E325, or Y101 enables a minor transamination activity for the substrate L-Ala.