Effect of a new non-cleavable substrate analog on wild-type and serine mutants in the signature sequence of adenylosuccinate lyase of Bacillus subtilis and Homo sapiens
Effect of a new non-cleavable substrate analog on wild-type and serine mutants in the signature sequence of adenylosuccinate lyase of Bacillus subtilis and Homo sapiens
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DOI：
10.1110/ps.034777.108
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发表时间：
2008-07-01
期刊：
影响因子：
8
通讯作者：
Colman, Roberta F.
中科院分区：
文献类型：
--
作者：
Sivendran, Sharmila;Colman, Roberta F.
腺苷酸琥珀酸裂解酶（ASL）催化嘌呤生物合成中的两个β-消除反应，导致两种底物是否占据相同或不同的活性位点的问题。枯草芽孢杆菌和人ASL与一种新底物类似物，腺苷膦酰基丁酸，2 '（3'），5 '-二磷酸（APBADP）的动力学研究表明，它作为一种竞争性抑制剂相对于任何一种底物（K-I类似于0.1 μ M），表明这两种底物占据相同的活性位点。结合研究表明，B.枯草芽孢杆菌和人ASL每摩尔酶四聚体结合高达4摩尔APBADP，并且两种酶都表现出协同性：对B呈阴性。枯草杆菌ASL和人ASL阳性。突变体B。枯草芽孢杆菌ASL，与以前确定为关键的催化残基的替代，结合底物类似物类似于野生型ASL。在ASL的柔性环中的两个丝氨酸已经被提出在催化中起作用，因为它们在大肠杆菌ASL的晶体结构中靠近底物。我们现在已经将B中相应的丝氨酸突变为丙氨酸。枯草芽孢杆菌和人ASL，以评估其参与酶功能。动力学数据表明人Ser（289）和B.枯草杆菌Ser（262）和Ser（263）是催化所必需的，而这些Ser突变体结合APBADP的能力表明它们对底物亲和力没有贡献。虽然这些丝氨酸在与底物或产物复合的人腺苷酸琥珀酸裂解酶（PDB #2 VD 6）的晶体结构中不可见，但它们可能与活性位点相互作用。
Adenylosuccinate lyase (ASL) catalyzes two beta-elimination reactions in purine biosynthesis, leading to the question of whether the two substrates occupy the same or different active sites. Kinetic studies of Bacillus subtilis and human ASL with a new substrate analog, adenosine phosphonobutyric acid, 2'(3'), 5'-diphosphate (APBADP), show that it acts as a competitive inhibitor with respect to either substrate (K-I similar to 0.1 mu M), indicating that the two substrates occupy the same active site. Binding studies show that both the B. subtilis and human ASLs bind up to 4 mol of APBADP per mole of enzyme tetramer and that both enzymes exhibit cooperativity: negative for B. subtilis ASL and positive for human ASL. Mutant B. subtilis ASLs, with replacements for residues previously identified as critical for catalysis, bind the substrate analog similarly to wild-type ASL. Two serines in a flexible loop of ASL have been proposed to play roles in catalysis because they are close to the substrate in the crystal structure of Escherichia coli ASL. We have now mutated the corresponding serines to alanines in B. subtilis and human ASL to evaluate their involvement in enzyme function. Kinetic data reveal that human Ser(289) and B. subtilis Ser(262) and Ser(263) are essential for catalysis, while the ability of these Ser mutants to bind APBADP suggests that they do not contribute to substrate affinity. Although these serines are not visible in the crystal structure of human adenylosuccinate lyase complexed with substrate or products (PDB # 2VD6), they may be interacting with the active sites.