Purification and characterization of cytidine 5'-triphosphate:cytidine 5'-monophosphate-3-deoxy-D-manno-octulosonate cytidylyltransferase.

Abstract

CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyltransferase (CMP-KDO synthetase) was purified 2300-fold from frozen Escherichia coli B cells. The enzyme catalyzed the formation of CMP-KDO (3-deoxy-D-manno-octulosonate), a very labile product, from CTP and KDO. No other sugar tested could replace KDO as an alternate substrate. UTP at pH 9.5 and dCTP at pH 8.0 and 9.5 could be used as alternate substrates in place of CTP. CMP-KDO synthetase required Mg2+ at a concentration of 10.0 mM for optimal activity. The pH optimum was determined to be between 9.6-9.3 in tris(hydroxymethyl)aminomethane-acetate or sodium-glycine buffer. This enzyme had an isoelectric point between pH 4.15-4.4 and appeared to be a single polypeptide chain with a MW of 36,000-40,000. The apparent Km values for CTP and KDO in the presence of 10.0 mM Mg2+ were determined to be 2.0 .times. 10-4 and 2.9 .times. 10-4 M, respectively, at pH 9.5. UTP and dCTP had apparent Km values of 8.8 .times. 10-4 and 3.4 .times. 10-4 M, respectively, at pH 9.5.