Kinetic properties of the acylneuraminate cytidylyltransferase from Pasteurella haemolytica A2
Kinetic properties of the acylneuraminate cytidylyltransferase from Pasteurella haemolytica A2
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DOI：
10.1042/bj3580585
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发表时间：
2001-09-15
影响因子：
4.1
通讯作者：
Reglero, A
中科院分区：
文献类型：
--
作者：
Bravo, IG;Barrallo, S;Reglero, A
神经侵袭性和引起败血症的病原体通常表现出参与侵袭行为的聚唾液酸胶囊。 N-乙酰神经氨酸（NeuAc）是聚唾液酸的基本单体。激活形式 CMP-Neu5Ac 由酰基神经氨酸胞苷酰转移酶 (ACT; EC 2.7.7.43) 合成。我们已经从溶血巴氏杆菌 A2 中纯化了这种酶，使其具有明显的同质性（522 倍）。该蛋白质在 SDS/PAGE 上表现均匀，为 43 kDa 带，大小与大肠杆菌、小牛、小鼠和大鼠的带相似。粗裂解物的比活性显示出文献中引用的最高值之一（153 m 单位/毫克）。我们通过使用归一化绘图前提研究了酶的稳态动力学机制。催化通过 Ping Pong Bi Bi 机制进行，CTP 作为第一个底物，CMP-NeuAc 作为最后的产物。 CTP 的真实 K-m 值为 1.77 mM，NeuAc 的真实 K-m 值为 1.82 mM。核苷酸CDP、UTP、UDP和TTP以及修饰的唾液酸N-羟乙酰神经氨酸也是ACT活性的底物。该酶通过与第二个胞苷基结合位点结合而被胞苷核苷酸抑制。对于具有长磷酸尾的核苷酸，这种抑制作用更大，真正的抑制剂是底物 CTP。在生理浓度下，ATP 是 ACT 活性的激活剂，AMP 是 ACT 活性的抑制剂。活化的糖 UDP-N-乙酰氨基葡萄糖充当抑制剂，因此表明肽聚糖和聚唾液酸途径的交叉调节。我们的研究结果为唾液酸激活的本质提供了新的机制见解，并为研究封装细菌的发病机制提出了新的靶标。
Neuroinvasive and septicaemia-causing pathogens often display a polysialic acid capsule that is involved in invasive behaviour. N-Acetylneuraminic acid (NeuAc) is the basic monomer of polysialic acid. The activated form, CMP-Neu5Ac, is synthesized by the acylneuraminate cytidylyltransferase (ACT; EC 2.7.7.43). We have purified this enzyme from Pasteurella haemolytica A2 to apparent homogeneity (522-fold). The protein behaved homogeneously on SDS/PAGE as a 43 kDa band, a size similar to that of Escherichia coli, calf, mouse and rat. Specific activity in crude lysate displayed one of the highest values cited in the literature (153 m-units/mg). We have studied the steady-state kinetic mechanism of the enzyme by using normalized plot premises. The catalysis proceeds through a Ping Pong Bi Bi mechanism, with CTP as the first substrate and CMP-NeuAc as the last product. The true K-m values were 1.77 mM for CTP and 1.82 mM for NeuAc. The nucleotides CDP, UTP, UDP and TTP, and the modified sialic acid N-glycolylneuraminic acid were also substrates of the ACT activity. The enzyme is inhibited by cytidine nucleotides through binding to a second cytidyl-binding site. This inhibition is greater with nucleotides that display a long phosphate tail, and the genuine inhibitor is the substrate CTP. At physiological concentrations, ATP is an activator, and AMP an inhibitor, of the ACT activity. The activated sugar UDP-N-acetylglucosamine acts as an inhibitor, thus suggesting cross-regulation of the peptidoglycan and polysialic acid pathways. Our findings provide new mechanistic insights into the nature of sialic acid activation and suggest new targets for the approach to the pathogenesis of encapsulated bacteria.