Department of Biology, Carleton University, Ottawa K1S 5B6, Canada.
Protein Sci. 2010 Mar;19(3):383-91. doi: 10.1002/pro.308.
Cystathionine beta-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(m) (L-Cth) for the beta-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 approximately 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 alpha-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(m) (L-Cth) 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.
胱硫醚β-裂合酶（CBL）催化 L-胱硫醚（L-Cth）水解生成 L-同型半胱氨酸、丙酮酸和氨。构建了一系列大肠杆菌 CBL（eCBL）的活性位点突变体，以研究残基 R58、R59、D116、W340 和 R372 在催化和氨基乙氧基乙烯基甘氨酸（AVG）抑制中的作用。这些突变对β消除反应的 k(cat)/K(m)（L-Cth）的影响范围从 D116A 和 D116N 的仅降低 3 倍到 R372L 和 R372A 突变体的 6 个数量级。这些残基对 L-Cth 水解的重要性顺序为：R372＞R58＞W340≈R59＞D116。L-Cth 水解的动力学参数与 AVG 抑制 eCBL 的动力学参数的比较表明，残基 R58 束缚了底物的远端羧酸盐基团，并证实了残基 W340 和 R372 与α-羧酸盐部分相互作用。R58K 和 R58A 突变体的 k(cat)/K(m)（L-Cth）与 pH 关系曲线的酸性臂的 pK(a)增加和碱性臂的 pK(a)降低，分别支持该残基在调节活性位点残基的 pK(a)方面的作用。
