Studies on the Cysteinyl-tRNA Synthetase from Rat Liver

鼠肝半胱胺酸活化酶之純化與特性研究

潘福(F. Pan) ； 白壽雄(S. H. Pai) ； 李賢雄(H. H. Lee) ； 康菱(L. Kang)

《Journal of Medical Sciences》 2卷4期 (1978/07) Pp. 747-759

摘要

由鼠肝純化蛋白質合成所需之半胱胺酸活化酶，成品純度可達99%以上，酶活性總收率約23%。利用磷酸鈣膠層析，該酶活性分成可以相互轉變之三部份（CRS-1，CRS-2，CRS-3）。實驗結果顯示三者係由不同數目之相同胜鏈單體所構成，各單體之分子量約為十二萬，三者之結構可分別以α, α2及α3（或α4）代表之。以純化之α2及α3的分別作為抗原製備抗體，利用膠片雙向擴散法與酶活性抑制試驗證明，兩種同功酶之抗原性近似而不相同，符合兩者係聚合性同功酶之結論。宜驗結果並顯示，兩種同功酶之存在，並非純化過程之人為結果。該酶分子之抗原性部位與催化活性部位並不相同。以各種生物來源之半胱胺酸活化酶免疫化學性質之比較研究，充分顯示生物進化與酶結構之關係。由基質特異性與動力學性質研究結果，證明該酶之催化部位，對基質之化學結構，具有最高度之選擇性。半胱胺酸與酶結合之主要部位，是彼此距離適當之硫醇基典L-胺基；羧基則非結合所必需。至於三磷酸腺苷與酶之有效結合，同時需要腺苷部份及整個三磷酸基。利用各種競爭性抑制劑研究結果顯示，在半胱胺酸活化反應中，基質與酶之結合屬於連續順序型，而以三磷酸腺苷為先行基質。2'-去氧三磷酸腺苷之抑制作用屬於拋物線形，顯示該酶分子至少具有兩個能與腺苷酸結合之部位。利用半胱胺醯腺苷酸生成反應，以活性中心滴定法，測定每分子CRS-2含有兩個活性中心。

並列摘要

Cysteinyl-tRNA synthetase (EC 6. 1. 1. 16) has been purified from rat liver and resolved into three active forms (Fractions CRS-1, CRS-2 and CRS-3). CRS-2 was homogeneous by polyacrylamide gel electrophoresis, while CRS-3 gave two active bands with mobilities corresponding to those of CRS-1 and CRS-2. Molecular weight determination by various methods indicate a dimeric structure of the type as for CRS-2, composed of two probably identical subunits of about 120,000 daltons. Stoichiometric study of cysteinyl adenylate formation indicates that CRS-2 has two active sites per molecule. Available evidence also suggests that CRS-1 and CRS-3 are α and α3 (or α4), respectively. Antisera against CRS-2 and CRS-3 were prepared and used in the immune-diffusion and immunotitration studies to assess the relationship of multiple forms of the enzyme. They were found to be closely related but nonidentical immunologically. The antisera inhibited both the cysteine activation and overall reaction, with the effect on the overall reaction being more severe than on the activation reaction. The antigenic region of the enzyme molecules appears to be different from the catalytic site. The cross-reaction of cysteinyl-tRNA synthetase from representative vertebrates, but not the yeast enzyme, with the antibody against the rat liver enzyme suggests a certain structural homology among the homologous enzymes.The substrate specificity and kinetic properties of the purified enzyme were investigated in the ATP-PP1 exchange reaction. L-Cysteine, ATP and PP1 were all inhibitory at high concentrations. The apparent Km values determined for these substrates with CRS-2 were 0.03, 1.8 and 0.16 mM, respectively. We found no significant difference in the Michaelis constants when CRS-1 or CRS-3 was used in place of CRS-2. None of the various analogues tested could replace L-cysteine or ATP as a substrate. Our data indicate that the L-amino and thiol groups at a proper distance are the major binding loci for cysteine, and that both the adenosine and entire triphosphate moieties are required for the effective binding of ATP to the active site of the enzyme. The inhibition patterns obtained with cysteamine, S-methyl-L-cysteine, 2'-deoxy-ATP and sodium tripolyphosphate are consistent with a sequential ordered mechanism of substrate binding, with ATP as the leading substrate. The kinetic data also suggest the involvement of at least two binding sites with different relative affinities for the nucleotide substrate and inhibitors.