C-13 NMR INVESTIGATION OF THE ANOMERIC SPECIFICITY OF CMP-N-ACETYLNEURAMINIC ACID SYNTHETASE FROM ESCHERICHIA-COLI

被引：27

作者：

AMBROSE, MG ^{[1
]}

FREESE, SJ ^{[1
]}

REINHOLD, MS ^{[1
]}

WARNER, TG ^{[1
]}

VANN, WF ^{[1
]}

机构：

[1] US FDA,CTR BIOL EVALUAT & RES,BACTERIAL POLYSACCHARIDES LAB,8800 ROCKVILLE PIKE,BETHESDA,MD 20892

来源：

BIOCHEMISTRY | 1992年 / 31卷 / 03期

关键词：

D O I：

10.1021/bi00118a019

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

The anomeric specificity of Escherichia coli CMP-N-acetylneuraminic acid (CMP-NeuAc) synthetase was investigated by NMR using C-13-labeled N-acetylneuraminic acid (NeuAc). Consumption of the beta-anomer of [2-C-13]N-acetylneuraminic acid was observed upon addition of enzyme, with a concomitant appearance of an anomeric resonance for CMP-N-acetylneuraminic acid. Inhibition by substrate analogues confirms the importance of the anomeric center for interaction of substrate with the enzyme. The fate of the anomeric oxygen was determined in a similar manner using [2-C-13,(50 atom %)O-18]N-acetylneuraminic acid. An upfield shift of 1.5 Hz in the anomeric resonance of both the [C-13]NeuAc substrate and CMP-[C-13]NeuAc product was observed due to the O-18 substitution. This result implies conservation of the NeuAc oxygen. Results of steady-state kinetic analysis suggest a sequential-type mechanism and therefore no covalent intermediate. Thus, CMP-beta-NeuAc is probably formed by a direct transfer of the anomeric oxygen of beta-NeuAc to the alpha-phosphate of CTP.

引用

页码：775 / 780

页数：6

共 32 条

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