BIOSYNTHESIS OF CAPSULAR POLYSACCHARIDES OF PATHOGENIC BACTERIA

大肠杆菌CMP-NeuAc合成酶催化胞苷5'的形成 单磷酸-N-乙酰神经氨酸（CMP-NeuAc），来自N-乙酰神经氨酸 酸（NeuAc）和胞苷三磷酸（CTP）。 可用性和特异性 纯化的糖活化酶和糖基转移酶使这些 酶是合成生物相关物质的方便工具 低聚糖。 半胱氨酸在结构和功能中的作用 酶的催化性质已通过定点 诱变和化学修饰。 化学改性与 巯基特异性试剂DTDP表明，一个半胱氨酸残基是 参与催化作用，因为这种酶可以完全失活， 用DTDP滴定一个半胱氨酸残基。 酶可以被保护 在底物CTP存在下失活。 定点 诱变证明半胱氨酸129和半胱氨酸329不是 对于催化是必需的，因为两者都可以被选定的氨基取代 不完全丧失活性的酸。 用N- 乙基马来酰亚胺（NEM）证明半胱氨酸329和半胱氨酸129是 在室温下NEM不可接近。 半胱氨酸329可以是热的。 暴露于42 ℃下用NEM但半胱氨酸129进行的化学修饰 无法用NEM进行化学修饰。 我们已经测序了 来自脑膜炎球菌B群的CMP NeuAc合成酶的氨基末端， 合成了同样的2-8 NeuAc E. coliK1。 N末端 序列进行了比较，并获得了共识。 精氨酸和赖氨酸 残基被鉴定为比较序列中的共同氨基酸。 我们在E.大肠杆菌CMP NeuAc合成酶转化为甘氨酸， 普通精氨酸的突变完全灭活CMP NeuAc 合成酶 精氨酸变成丙氨酸会导致酶失活， 但是赖氨酸的改变使比活性恢复到野生型值。 在不同的pH值下进行Km测定，以确定参与 精氨酸的催化作用。

Escherichia coli CMP-NeuAc synthetase catalyses formation of cytidine 5' monophospho-N-acetylneuraminic acid (CMP-NeuAc) from N-acetylneuraminic acid (NeuAc) and cytidine triphosphate (CTP). Availability and specificity of purified sugar activating enzymes and glycosyltransferases make these enzymes convenient tools for synthesis of biologically relevant oligosaccharides. The role of the cysteines in the structural and catalytic properties of the enzyme has been examined by site directed mutagenesis and chemical modification. Chemical modification with the sulfhydro specific reagent DTDP suggests that one cysteine residue is involved in catalysis, since that enzyme can be completely inactivated by titration of one cysteine residue with DTDP. The enzyme can be protected from inactivation in the presence of the substrate CTP. Site directed mutagenesis demonstrates that cysteine 129 and cysteine 329 are not essential for catalysis, since both can be substituted by selected amino acids without complete loss of activity. Chemical inactivation with N- ethylmaleimide (NEM) demonstrates that cysteine 329 and cysteine 129 are inaccessible to NEM at room temperature. Cysteine 329 can be thermally exposed to chemical modification with NEM at 42 degree C but cysteine 129 is inaccessible to chemical modification with NEM. We have sequenced the amino terminus of the CMP NeuAc synthetase from meningococcus group B which synthesizes the same a 2-8 NeuAc capsule of E. coli K 1. The N terminal sequences were compared and a concensus obtained. Arginine and lysine residues were identified as common amino acids in the compared sequences. We have mutated 14 arginines in E. coli CMP NeuAc synthetase to Glycine and mutation of the common arginine completely inactivates CMP NeuAc synthetase. Changing arginine to alanine results in an inactivated enzyme, but a change to lysine restores the specific activity to wild type values. Km determinations are being done at different pHs to determine involvement of arginine in catalysis.