Padgette S R, Huynh Q K, Aykent S, Sammons R D, Sikorski J A, Kishore G M
Plant Molecular Biology Group, Monsanto Company, Chesterfield, Missouri 63198.
J Biol Chem. 1988 Feb 5;263(4):1798-802.
Reaction of 5-enolpyruvylshikimate-3-phosphate synthase of Escherichia coli with the thiol reagent 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) leads to a modification of only 2 of the 6 cysteines of the enzyme, with a significant loss of its enzymatic activity. Under denaturing conditions, however, all 6 cysteines of 5-enolpyruvylshikimate-3-phosphate synthase react with DTNB, indicating the absence of disulfide bridges in the native protein. In the presence of shikimate 3-phosphate and glyphosate, only 1 of the 2 cysteines reacts with the reagent, with no loss of activity, suggesting that only 1 of these cysteines is at or near the active site of the enzyme. Cyanolysis of the DTNB-inactivated enzyme with KCN leads to elimination of 5-thio-2-nitrobenzoate, with formation of the thiocyano-enzyme. The thiocyano-enzyme is fully active; it exhibits a small increase in its I50 for glyphosate (6-fold) and apparent Km for phosphoenolpyruvate (4-fold) compared to the unmodified enzyme. Its apparent Km for shikimate 3-phosphate is, however, unaltered. These results clearly establish the nonessentiality of the active site-reactive cysteine of E. coli 5-enolpyruvylshikimate-3-phosphate synthase for either catalysis or substrate binding. Perturbations in the kinetic constants for phosphoenolpyruvate and glyphosate suggest that the cysteine thiol is proximal to the binding site for these ligands. By N-[14C]ethylmaleimide labeling, tryptic mapping, and N-terminal sequencing, the 2 reactive cysteines have been identified as Cys408 and Cys288. The cysteine residue protected by glyphosate and shikimate 3-phosphate from its reaction with DTNB was found to be Cys408.
大肠杆菌5-烯醇丙酮酸莽草酸-3-磷酸合酶与硫醇试剂5,5'-二硫代双（2-硝基苯甲酸）（DTNB）反应，仅使该酶的6个半胱氨酸中的2个发生修饰，其酶活性显著丧失。然而，在变性条件下，5-烯醇丙酮酸莽草酸-3-磷酸合酶的所有6个半胱氨酸都与DTNB反应，这表明天然蛋白质中不存在二硫键。在莽草酸3-磷酸和草甘膦存在的情况下，2个半胱氨酸中只有1个与试剂反应，且活性没有丧失，这表明这些半胱氨酸中只有1个位于酶的活性位点或其附近。用KCN对DTNB失活的酶进行氰解，导致5-硫代-2-硝基苯甲酸消除，形成硫氰基酶。硫氰基酶具有完全活性；与未修饰的酶相比，它对草甘膦的I50略有增加（6倍），对磷酸烯醇丙酮酸的表观Km增加（4倍）。然而，它对莽草酸3-磷酸的表观Km未改变。这些结果清楚地表明，大肠杆菌5-烯醇丙酮酸莽草酸-3-磷酸合酶的活性位点反应性半胱氨酸对于催化或底物结合并非必需。磷酸烯醇丙酮酸和草甘膦动力学常数的扰动表明，半胱氨酸硫醇靠近这些配体的结合位点。通过N-[14C]乙基马来酰亚胺标记、胰蛋白酶图谱分析和N端测序，已确定2个反应性半胱氨酸为Cys408和Cys288。发现受草甘膦和莽草酸3-磷酸保护而不与DTNB反应的半胱氨酸残基是Cys408。
