Storici P, Capitani G, Müller R, Schirmer T, Jansonius J N
Division of Structural Biology Biozentrum, University of Basel, Klingelbergstrasse 70, Basel, CH-4056, Switzerland.
J Mol Biol. 1999 Jan 8;285(1):297-309. doi: 10.1006/jmbi.1998.2289.
Ornithine aminotransferase (l-ornithine:2-oxoacid delta-aminotransferase; EC 2.6.1.13), a pyridoxal-5'-phosphate-dependent mitochondrial enzyme controls the l-ornithine level in tissues by catalyzing the transfer of the delta-amino group of l-ornithine to 2-oxoglutarate, producing l-glutamate- gamma-semialdehyde and l-glutamate. (2S, 5S)-5-Fluoromethylornithine is the only inhibitor exclusively specific for ornithine aminotransferase known to date. Both in vitro and in vivo, it blocks the enzyme by a suicide reaction leading to a covalent adduct with the cofactor. The crystal structure of the enzyme-inhibitor complex was solved at a resolution of 1.95 A. No significant conformational changes compared with the native enzyme structure were observed. The structure reveals the atomic details of the cofactor-inhibitor adduct and its interactions with the active site of the enzyme. The main residues responsible for specific binding of the inhibitor are Arg180, which forms a strong salt bridge with the alpha-carboxylate and Tyr55, which is involved in a short hydrogen bond with the alpha-amino group. The experimental observation that in the racemic mixture, (2S, 5S)-5-fluoromethylornithine is exclusively responsible for the enzyme inhibition can be explained on the basis of the active site topology. Model building studies strongly suggest that the natural substrate l-ornithine, in its external aldimine adduct with the enzyme, makes use of the same recognition site as the inhibitor. It is proposed that the neutralization of the active site Arg413 by a salt bridge with Glu235 also plays an important role in productive binding of both 5-fluoromethylornithine and l-ornithine. Arg180 and Arg413 are believed to be instrumental in recognition of l-glutamate, by binding its gamma and alpha-carboxylate groups, respectively. This requires a different side-chain conformation of Glu235. Lys292 is the only obvious candidate for catalyzing the rate-limiting proton transfer steps in the transamination reaction.
鸟氨酸转氨酶（L-鸟氨酸：2-氧代酸δ-转氨酶；EC 2.6.1.13）是一种依赖磷酸吡哆醛的线粒体酶，通过催化L-鸟氨酸的δ-氨基转移至2-氧代戊二酸，生成L-谷氨酸-γ-半醛和L-谷氨酸，从而控制组织中的L-鸟氨酸水平。（2S，5S）-5-氟甲基鸟氨酸是迄今为止已知的唯一一种对鸟氨酸转氨酶具有特异性的抑制剂。在体外和体内，它都通过自杀反应阻断该酶，导致与辅因子形成共价加合物。酶-抑制剂复合物的晶体结构以1.95 Å的分辨率解析。与天然酶结构相比，未观察到明显的构象变化。该结构揭示了辅因子-抑制剂加合物的原子细节及其与酶活性位点的相互作用。负责抑制剂特异性结合的主要残基是与α-羧酸盐形成强盐桥的Arg180和与α-氨基形成短氢键的Tyr55。在外消旋混合物中，（2S，5S）-5-氟甲基鸟氨酸是唯一导致酶抑制的成分，这一实验观察结果可根据活性位点拓扑结构来解释。模型构建研究强烈表明，天然底物L-鸟氨酸在与酶形成的外部醛亚胺加合物中，利用了与抑制剂相同的识别位点。有人提出，与Glu235形成盐桥使活性位点Arg413中和，这在5-氟甲基鸟氨酸和L-鸟氨酸的有效结合中也起着重要作用。据信，Arg180和Arg413分别通过结合L-谷氨酸的γ-和α-羧酸盐基团，有助于识别L-谷氨酸。这需要Glu235具有不同的侧链构象。Lys292是催化转氨反应中限速质子转移步骤的唯一明显候选残基。
