Klem T J, Chen Y, Davisson V J
Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana 47907-1333.
J Bacteriol. 2001 Feb;183(3):989-96. doi: 10.1128/JB.182.3.989-996.2001.
A selection strategy has been developed to identify amino acid residues involved in subunit interactions that coordinate the two half-reactions catalyzed by glutamine amidotransferases. The protein structures known for this class of enzymes have revealed that ammonia is shuttled over long distances and that each amidotransferase evolved different molecular tunnels for this purpose. The heterodimeric Escherichia coli imidazole glycerol phosphate (IGP) synthase was probed to assess if residues in the substrate amination subunit (HisF) are critical for the glutaminase activity in the HisH subunit. The activity of the HisH subunit is dependent upon binding of the nucleotide substrate at the HisF active site. This regulatory function has been exploited as a biochemical selection of mutant HisF subunits that retain full activity with ammonia as a substrate but, when constituted as a holoenzyme with wild-type HisH, impair the glutamine-dependent activity of IGP synthase. The steady-state kinetic constants for these IGP synthases with HisF alleles showed three distinct effects depending upon the site of mutation. For example, mutation of the R5 residue has similar effects on the glutamine-dependent amidotransfer reaction; however, k(cat)/K(m) for the glutaminase half-reaction was increased 10-fold over that for the wild-type enzyme with nucleotide substrate. This site appears essential for coupling of the glutamine hydrolysis and ammonia transfer steps and is the first example of a site remote to the catalytic triad that modulates the process. The results are discussed in the context of recent X-ray crystal structures of glutamine amidotransferases that relate the glutamine binding and acceptor binding sites.
已开发出一种筛选策略，以鉴定参与亚基相互作用的氨基酸残基，这些相互作用协调了谷氨酰胺酰胺转移酶催化的两个半反应。这类酶已知的蛋白质结构表明，氨会远距离穿梭，并且每种酰胺转移酶为此进化出了不同的分子通道。对异源二聚体大肠杆菌咪唑甘油磷酸（IGP）合酶进行了研究，以评估底物胺化亚基（HisF）中的残基对于HisH亚基中的谷氨酰胺酶活性是否至关重要。HisH亚基的活性取决于核苷酸底物在HisF活性位点的结合。这种调节功能已被用作对突变HisF亚基的生化筛选，这些亚基以氨作为底物时保留全部活性，但当与野生型HisH组成全酶时，会损害IGP合酶的谷氨酰胺依赖性活性。这些带有HisF等位基因的IGP合酶的稳态动力学常数根据突变位点显示出三种不同的效应。例如，R5残基的突变对谷氨酰胺依赖性酰胺转移反应具有相似的影响；然而，谷氨酰胺酶半反应的k(cat)/K(m)比野生型酶与核苷酸底物的k(cat)/K(m)增加了10倍。该位点似乎对于谷氨酰胺水解和氨转移步骤的偶联至关重要，并且是远离催化三联体调节该过程的位点的首个例子。将结合谷氨酰胺酰胺转移酶的谷氨酰胺结合位点和受体结合位点的最新X射线晶体结构对结果进行讨论。
