Zapata G, Roller P P, Crowley J, Vann W F
Laboratory of Bacterial Polysaccharides, Center for Biologics Evaluation and Research, Bethesda, MD 20892.
Biochem J. 1993 Oct 15;295 ( Pt 2)(Pt 2):485-91. doi: 10.1042/bj2950485.
N-Acetylneuraminic acid cytidyltransferase (CMP-NeuAc synthase) of Escherichia coli K1 is sensitive to mercurials and has cysteine residues only at positions 129 and 329. The role of these residues in the catalytic activity and structure of the protein has been investigated by site-directed mutagenesis and chemical modification. The enzyme is inactivated by the thiol-specific reagent dithiodipyridine. Inactivation by this reagent is decreased in the presence of the nucleotide substrate CTP, suggesting that a thiol residue is at or near the active site. Site-directed mutagenesis of either residue Cys-129 to serine or Cys-329 to selected amino acids has minor effects on the specific activity of the enzyme, suggesting that cysteine is not essential for catalysis and that a disulphide bond is not an essential structural component. The limited reactivity of the enzyme to other thiol-blocking reagents suggests that its cysteine residues are partially exposed. The accessibility and role of the cysteine residues in enzyme structure were investigated by fluorescence, c.d. and denaturation studies of wild-type and mutant enzymes. The mutation of Cys-129 to serine makes the enzyme more sensitive to heat and chemical denaturation, but does not cause gross changes in the protein structure as judged by the c.d. spectrum. The mutant containing Ser-129 instead of Cys-129 had a complex denaturation pathway similar to that of wild-type E. coli K1 CMP-NeuAc synthase consisting of several partially denatured states. Cys-329 reacts more readily with N-[14C]ethylmaleimide when the enzyme is in a heat-induced relaxed state. Cys-129 is less reactive and is probably a buried residue.
大肠杆菌K1的N - 乙酰神经氨酸胞苷转移酶（CMP - NeuAc合酶）对汞制剂敏感，且仅在第129位和第329位含有半胱氨酸残基。通过定点诱变和化学修饰研究了这些残基在蛋白质催化活性和结构中的作用。该酶可被硫醇特异性试剂二硫代二吡啶灭活。在核苷酸底物CTP存在的情况下，该试剂引起的失活作用减弱，这表明硫醇残基位于活性位点或其附近。将残基Cys - 129定点突变为丝氨酸或将Cys - 329定点突变为选定的氨基酸对该酶的比活性影响较小，这表明半胱氨酸对于催化并非必不可少，且二硫键不是必需的结构成分。该酶对其他硫醇封闭试剂的反应性有限，表明其半胱氨酸残基部分暴露。通过对野生型和突变型酶进行荧光、圆二色光谱和变性研究，考察了半胱氨酸残基在酶结构中的可及性和作用。将Cys - 129突变为丝氨酸使该酶对热和化学变性更敏感，但根据圆二色光谱判断，蛋白质结构并未发生明显变化。含有Ser - 129而非Cys - 129的突变体具有与野生型大肠杆菌K1 CMP - NeuAc合酶相似的复杂变性途径，由几种部分变性状态组成。当酶处于热诱导的松弛状态时，Cys - 329与N - [14C]乙基马来酰亚胺的反应更易发生。Cys - 129的反应性较低，可能是一个埋藏残基。
