Harpel Mark R, Horiuchi Kurumi Y, Luo Ying, Shen Li, Jiang Wenjun, Nelson David J, Rogers Kelley C, Decicco Carl P, Copeland Robert A
Chemical Enzymology Department, Bristol-Myers Squibb Company, Experimental Station, Route 141 and Henry Clay Road, Wilmington, Delaware 19880, USA.
Biochemistry. 2002 May 21;41(20):6398-407. doi: 10.1021/bi012126u.
The absence of Gln-tRNA synthetase in certain bacteria necessitates an alternate pathway for the production of Gln-tRNA(Gln): misacylated Glu-tRNA(Gln) is transamidated by a Gln-dependent amidotransferase (Glu-AdT) via catalysis of Gln hydrolysis, ATP hydrolysis, activation of Glu-tRNA(Gln), and aminolysis of activated tRNA by Gln-derived NH(3). As observed for other Gln-coupled amidotransferases, substrate binding, Gln hydrolysis, and transamidation by Glu-AdT are tightly coordinated [Horiuchi, K. Y., Harpel, M. R., Shen, L., Luo, Y., Rogers, K. C., and Copeland, R. A. (2001) Biochemistry 40, 6450-6457]. However, Glu-AdT does not employ an active-site Cys nucleophile for Gln hydrolysis, as is common in all other glutaminases: some Glu-AdT lack Cys, but all contain a conserved Ser (Ser176 in the A subunit of Streptococcus pyogenes Glu-AdT) within a sequence signature motif of Ser-based amidases. Our current results with S. pyogenes Glu-AdT support this characterization of Glu-AdT as a Ser-based glutaminase. Slow-onset (approximately 50 M(-1) s(-1)), tight-binding (t(1/2) > 2.5 h for complex dissociation), Gln-competitive inhibition of the Glu-tRNA(Gln)/ATP-independent glutaminase activity of Glu-AdT by gamma-Glu boronic acid is consistent with engagement of a Ser nucleophile in the glutaminase active site. Conversion to rapidly reversible, yet still potent (K(i) = 73 nM) and Gln-competitive, inhibition under full transamidation conditions mirrors the coupling between Gln hydrolysis and aminolysis reactions during productive transamidation. Site-directed replacement of Ser176 by Ala abolishes glutaminase and Gln-dependent transamidase activities of Glu-AdT (>300-fold), but retains a wild-type level of NH(3)-dependent transamidation activity. These results demonstrate the essentiality of Ser176 for Gln hydrolysis, provide additional support for coordinated coupling of Gln hydrolysis and transamidase transition states during catalysis, and validate glutaminase-directed inhibition of Glu-AdT as a route for antimicrobial chemotherapy.
某些细菌中缺乏谷氨酰胺 - tRNA合成酶，这就需要一条替代途径来生成谷氨酰胺 - tRNA(Gln)：错误酰化的谷氨酸 - tRNA(Gln)通过谷氨酰胺依赖性酰胺转移酶（Glu - AdT）进行转酰胺作用，该过程经由谷氨酰胺水解、ATP水解、谷氨酸 - tRNA(Gln)的活化以及谷氨酰胺衍生的NH(3)对活化tRNA的氨解作用。正如其他谷氨酰胺偶联的酰胺转移酶所观察到的那样，底物结合、谷氨酰胺水解以及Glu - AdT的转酰胺作用紧密协调[堀内，K. Y.，哈佩尔，M. R.，沈，L.，罗，Y.，罗杰斯，K. C.，以及科普兰，R. A.（2001年）《生物化学》40卷，6450 - 6457页]。然而，Glu - AdT在谷氨酰胺水解过程中并不像所有其他谷氨酰胺酶那样使用活性位点的半胱氨酸亲核试剂：一些Glu - AdT缺乏半胱氨酸，但所有Glu - AdT在基于丝氨酸的酰胺酶的序列特征基序内都含有一个保守的丝氨酸（化脓性链球菌Glu - AdT的A亚基中的Ser176）。我们目前对化脓性链球菌Glu - AdT的研究结果支持了将Glu - AdT表征为基于丝氨酸的谷氨酰胺酶。γ - 谷氨酸硼酸对Glu - AdT的谷氨酸 - tRNA(Gln)/ATP非依赖性谷氨酰胺酶活性的抑制作用起效缓慢（约50 M(-1) s(-1)）、结合紧密（复合物解离的t(1/2) > 2.5小时）且具有谷氨酰胺竞争性，这与丝氨酸亲核试剂参与谷氨酰胺酶活性位点的作用一致。在完全转酰胺作用条件下转化为快速可逆但仍然强效（K(i) = 73 nM）且具有谷氨酰胺竞争性的抑制作用，反映了在有效转酰胺作用过程中谷氨酰胺水解与氨解反应之间的偶联。将Ser176定点替换为丙氨酸会消除Glu - AdT的谷氨酰胺酶和谷氨酰胺依赖性转酰胺酶活性（>300倍），但保留野生型水平的NH(3)依赖性转酰胺酶活性。这些结果证明了Ser176对于谷氨酰胺水解的必要性，为催化过程中谷氨酰胺水解和转酰胺酶过渡态的协调偶联提供了额外支持，并验证了以谷氨酰胺酶为导向抑制Glu - AdT作为抗菌化疗途径的有效性。
