Lei Y, Ploux O, Liu H W
Department of Chemistry, University of Minnesota, Minneapolis 55455, USA.
Biochemistry. 1995 Apr 11;34(14):4643-54. doi: 10.1021/bi00014a018.
CDP-6-deoxy-L-threo-D-glycero-4-hexulose 3-dehydrase (E1) purified from Yersinia pseudotuberculosis is a pyridoxamine 5'-phosphate (PMP) dependent iron-sulfur-containing enzyme which catalyzes the C-O bond cleavage at C-3 of its substrate leading to the formation of 3,6-dideoxyhexose. This enzyme is rapidly inactivated by diethyl pyrocarbonate (DEP) at pH 6.0 and 25 degrees C. The inactivation of E1 by DEP, which is reversible upon treatment of hydroxylamine, appears to be attributable solely to the modification of histidine residues. The fact that coincubation of E1 with its substrate gave almost total protection against DEP inactivation and that only one less histidine residue was modified in the presence of substrate strongly suggested that inactivation is due to the modification of only one reactive histidine residue which resides in or near the active site of E1 and is critical for E1's activity. Sequence alignment between the translated ascC (E1) gene and several representative pyridoxal 5'-phosphate (PLP)/PMP dependent enzymes revealed that three of the four invariant residues, glycine, aspartate, and arginine found in all other aminotransferases, are conserved in the E1 sequence (G169, D191, and R403). However, the highly conserved lysine is replaced by a histidine residue (H220) in E1. In order to test whether H220 plays an essential role in E1 catalysis, H220N mutant was constructed and the encoding protein was found to exhibit nearly identical physical characteristics as the wild-type E1. Interestingly, the mutant protein had lost most of its catalytic activity, and one less histidine residue was modified upon treatment of H220N-mutated protein with DEP. Such a single-point mutation also impaired E1's capability of catalyzing the solvent hydrogen exchange at C-4' position of the PMP coenzyme. Our findings strongly suggested that H220 is most likely the active-site base which abstracts the C-4' proton from the PMP-substrate adduct and initiates the catalysis. Furthermore, E1's preservation of other invariant residues found in many PLP/PMP dependent enzymes allowed a speculation of their roles in E1 catalysis.(ABSTRACT TRUNCATED AT 400 WORDS)
从假结核耶尔森氏菌中纯化得到的CDP-6-脱氧-L-苏式-D-甘油-4-己酮糖3-脱水酶（E1）是一种依赖磷酸吡哆胺5'-磷酸（PMP）的含铁硫酶，它催化底物C-3位的C-O键断裂，生成3,6-二脱氧己糖。该酶在pH 6.0和25℃条件下会被焦碳酸二乙酯（DEP）迅速灭活。DEP对E1的灭活作用在羟胺处理后是可逆的，这似乎完全归因于组氨酸残基的修饰。E1与其底物共同孵育能几乎完全保护其免受DEP灭活，且在有底物存在时只有一个组氨酸残基被修饰，这一事实强烈表明，灭活是由于仅一个位于E1活性位点内或附近的活性组氨酸残基被修饰，而该残基对E1的活性至关重要。对翻译后的ascC（E1）基因与几种代表性的磷酸吡哆醛5'-磷酸（PLP）/PMP依赖性酶进行序列比对发现，在所有其他转氨酶中发现的四个不变残基中的三个，即甘氨酸、天冬氨酸和精氨酸，在E1序列中是保守的（G169、D191和R403）。然而，高度保守的赖氨酸在E1中被一个组氨酸残基（H220）取代。为了测试H220在E1催化中是否起关键作用，构建了H220N突变体，发现编码的蛋白质与野生型E1具有几乎相同的物理特性。有趣的是，突变蛋白几乎失去了所有催化活性，用DEP处理H220N突变蛋白后，被修饰的组氨酸残基少了一个。这样的单点突变也损害了E1催化PMP辅酶C-4'位溶剂氢交换的能力。我们的研究结果强烈表明，H220很可能是从PMP-底物加合物中夺取C-4'质子并启动催化的活性位点碱基。此外，E1保留了许多PLP/PMP依赖性酶中发现的其他不变残基，这使得人们可以推测它们在E1催化中的作用。（摘要截短至400字）
