Kinetic and crystallographic studies of Escherichia coli UDP-N-acetylmuramate:L-alanine ligase
Kinetic and crystallographic studies of Escherichia coli UDP-N-acetylmuramate:L-alanine ligase
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DOI：
10.1002/pro.5560051219
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发表时间：
1996-12-01
期刊：
影响因子：
8
通讯作者：
Villafranca, JJ
中科院分区：
文献类型：
--
作者：
Emanuele, JJ;Jin, HY;Villafranca, JJ
尿苷二磷酸-N-乙酰胞壁酸:L-丙氨酸连接酶（EC 6.3.2.8、UNAM:L-Ala 连接酶或 MurC 基因产物）催化第一个氨基酸与肽聚糖前体的糖部分的 ATP 依赖性连接。这是革兰氏阳性和革兰氏阴性细菌细胞壁生物合成的重要步骤。已经建立了初始速度研究的最佳测定条件。进行稳态测定以确定各种参数对酶活性的影响。研究的因素包括：阳离子特异性、离子强度、缓冲液成分和 pH 值。在 37 摄氏度和 pH 8.0 下，k(cat) 等于 980 +/- 40 min(-1)，而 ATP、UNAM 和 L-丙氨酸的 K-m 值分别为 130 +/- 10、44 +/- 3 和 48 +/- 6 muM。在测试的金属中，只有锰、镁和钴能够支持活性。氯化钠、氯化钾、氯化铵和硫酸铵在浓度高达 75 mM 时对活性没有影响。该酶在适当的缓冲液中足够稳定，可以在 5.6 至 10.1 的 pH 范围内进行测定，获得了三种底物的 V-max/K-m 和 V-max 的 pH 曲线。酶的结晶实验产生了两种晶型。其中一种通过 X 射线衍射表征为单斜晶体，空间群 C2，晶胞尺寸 a = 189.6，b = 92.1，c = 75.2 埃，β = 105 度，每个不对称单元有两个 54 kDa 分子。人们发现，在缺乏 L-丙氨酸的情况下，该酶会水解 ATP。这种 L-丙氨酸独立活性取决于 ATP 和 UNAM 的浓度；该活性的 k(cat) 小于在 L-丙氨酸饱和水平存在下测得的生物合成活性的 4%。许多经测试的 L-丙氨酸类似物都显示出刺激 ATP 水解。通过 HPLC 和质谱分析，许多 L-丙氨酸类似物产生了新产品。所有作为抑制剂测试的 L-丙氨酸类似物都与 L-丙氨酸具有竞争性。
Uridine diphosphate-N-acetylmuramate:L-alanine ligase (EC 6.3.2.8, UNAM:L-Ala ligase or MurC gene product) catalyzes the ATP-dependent ligation of the first amino acid to the sugar moiety of the peptidoglycan precursor. This is an essential step in cell wall biosynthesis for both gram-positive and gram-negative bacteria. Optimal assay conditions for initial velocity studies have been established. Steady-state assays were carried out to determine the effect of various parameters on enzyme activity. Factors studied included: cation specificity, ionic strength, buffer composition and pH. At 37 degrees C and pH 8.0, k(cat) was equal to 980 +/- 40 min(-1), while K-m values for ATP, UNAM, and L-alanine were, 130 +/- 10, 44 +/- 3, and 48 +/- 6 mu M, respectively. Of the metals tested only Mn, Mg, and Co were able to support activity. Sodium chloride, potassium chloride, ammonium chloride, and ammonium sulfate had no effect on activity up to 75 mM levels. The enzyme, in appropriate buffer, was stable enough to be assayed over the pH range of 5.6 to 10.1, pH profiles of V-max/K-m for the three substrates and of V-max were obtained. Crystallization experiments with the enzyme produced two crystal forms. One of these has been characterized by X-ray diffraction as monoclinic, space group C2, with cell dimensions a = 189.6, b = 92.1, c = 75.2 Angstrom, beta = 105 degrees, and two 54 kDa molecules per asymmetric unit. It was discovered that the enzyme will hydrolyze ATP in the absence of L-alanine. This L-alanine independent activity is dependent upon the concentrations of both ATP and UNAM; k(cat) for this activity is less than 4% of the biosynthetic activity measured in the presence of saturating levels of L-alanine. Numerous L-alanine analogs tested were shown to stimulate ATP hydrolysis. A number of these L-alanine analogs produced novel products as accessed by HPLC and mass spectral analysis. All of the L-alanine analogs tested as inhibitors were competitive versus L-alanine.