Grimek T L, Holden H, Rayment I, Escalante-Semerena J C
Department of Bacteriology, University of Wisconsin--Madison, Madison, Wisconsin 53726-4087, USA.
J Bacteriol. 2003 Aug;185(16):4837-43. doi: 10.1128/JB.185.16.4837-4843.2003.
The prpB gene of Salmonella enterica serovar Typhimurium LT2 encodes a protein with 2-methylisocitrate (2-MIC) lyase activity, which cleaves 2-MIC into pyruvate and succinate during the conversion of propionate to pyruvate via the 2-methylcitric acid cycle. This paper reports the isolation and kinetic characterization of wild-type and five mutant PrpB proteins. Wild-type PrpB protein had a molecular mass of approximately 32 kDa per subunit, and the biologically active enzyme was comprised of four subunits. Optimal 2-MIC lyase activity was measured at pH 7.5 and 50 degrees C, and the reaction required Mg(2+) ions; equimolar concentrations of Mn(2+) ions were a poor substitute for Mg(2+) (28% specific activity). Dithiothreitol (DTT) or reduced glutathione (GSH) was required for optimal activity; the role of DTT or GSH was apparently not to reduce disulfide bonds, since the disulfide-specific reducing agent Tris(2-carboxyethyl)phosphine hydrochloride failed to substitute for DTT or GSH. The K(m) of PrpB for 2-MIC was measured at 19 micro M, with a k(cat) of 105 s(-1). Mutations in the prpB gene were introduced by site-directed mutagenesis based on the active-site residues deemed important for catalysis in the closely related phosphoenolpyruvate mutase and isocitrate lyase enzymes. Residues D58, K121, C123, and H125 of PrpB were changed to alanine, and residue R122 was changed to lysine. Nondenaturing polyacrylamide gel electrophoresis indicated that all mutant PrpB proteins retained the same oligomeric state of the wild-type enzyme, which is known to form tetramers. The PrpB(K121A), PrpB(H125A), and PrpB(R122K) mutant proteins formed enzymes that had 1,050-, 750-, and 2-fold decreases in k(cat) for 2-MIC lyase activity, respectively. The PrpB(D58A) and PrpB(C123A) proteins formed tetramers that displayed no detectable 2-MIC lyase activity indicating that both of these residues are essential for catalysis. Based on the proposed mechanism of the closely related isocitrate lyases, PrpB residue C123 is proposed to serve as the active site base, and residue D58 is critical for the coordination of a required Mg(2+) ion.
鼠伤寒沙门氏菌LT2血清型的prpB基因编码一种具有2-甲基异柠檬酸（2-MIC）裂解酶活性的蛋白质，该酶在丙酸通过2-甲基柠檬酸循环转化为丙酮酸的过程中，将2-MIC裂解为丙酮酸和琥珀酸。本文报道了野生型和五种突变型PrpB蛋白的分离及动力学特性。野生型PrpB蛋白每个亚基的分子量约为32 kDa，生物活性酶由四个亚基组成。在pH 7.5和50℃下测得最佳2-MIC裂解酶活性，该反应需要Mg(2+)离子；等摩尔浓度的Mn(2+)离子是Mg(2+)的较差替代物（比活性为28%）。最佳活性需要二硫苏糖醇（DTT）或还原型谷胱甘肽（GSH）；DTT或GSH的作用显然不是还原二硫键，因为二硫键特异性还原剂盐酸三（2-羧乙基）膦不能替代DTT或GSH。PrpB对2-MIC的K(m)值为19 μM，k(cat)为105 s(-1)。基于对密切相关的磷酸烯醇丙酮酸变位酶和异柠檬酸裂解酶中对催化重要的活性位点残基，通过定点诱变在prpB基因中引入突变。PrpB的D58、K121、C123和H125残基被替换为丙氨酸，R122残基被替换为赖氨酸。非变性聚丙烯酰胺凝胶电泳表明，所有突变型PrpB蛋白都保持了野生型酶相同的寡聚状态，已知野生型酶形成四聚体。PrpB(K121A)、PrpB(H125A)和PrpB(R122K)突变蛋白形成的酶对2-MIC裂解酶活性的k(cat)分别降低了1050倍、750倍和2倍。PrpB(D58A)和PrpB(C123A)蛋白形成的四聚体没有可检测到的2-MIC裂解酶活性，表明这两个残基对催化都是必不可少的。基于密切相关的异柠檬酸裂解酶的推测机制，PrpB残基C123被认为是活性位点碱基，残基D58对所需Mg(2+)离子的配位至关重要。
