Arnone M I, Birolo L, Giamberini M, Cubellis M V, Nitti G, Sannia G, Marino G
Dipartimento di Chimica Organica e Biologica, Università di Napoli, Italy.
Eur J Biochem. 1992 Mar 15;204(3):1183-9. doi: 10.1111/j.1432-1033.1992.tb16745.x.
The analysis of conformational transitions using limited proteolysis was carried out on a hyperthermophilic aspartate aminotransferase isolated from the archaebacterium Sulfolobus solfataricus, in comparison with pig cytosolic aspartate aminotransferase, a thoroughly studied mesophilic aminotransferase which shares about 15% similarity with the archaebacterial protein. Aspartate aminotransferase from S. solfataricus is cleaved at residue 28 by thermolysin and residues 32 and 33 by trypsin; analogously, pig heart cytosolic aspartate aminotransferase is cleaved at residues 19 and 25 [Iriarte, A., Hubert, E., Kraft, K. & Martinez-Carrion, M. (1984) J. Biol. Chem. 259, 723-728] by trypsin. In the case of aspartate aminotransferase from S. solfataricus, proteolytic cleavages also result in transaminase inactivation thus indicating that both enzymes, although evolutionarily distinct, possess a region involved in catalysis and well exposed to proteases which is similarly positioned in their primary structure. It has been reported that the binding of substrates induces a conformational transition in aspartate aminotransferases and protects the enzymes against proteolysis [Gehring, H. (1985) in Transaminases (Christen, P. & Metzler, D. E., eds) pp. 323-326, John Wiley & Sons, New York]. Aspartate aminotransferase from S. solfataricus is protected against proteolysis by substrates, but only at high temperatures (greater than 60 degrees C). To explain this behaviour, the kinetics of inactivation caused by thermolysin were measured in the temperature range 25-75 degrees C. The Arrhenius plot of the proteolytic kinetic constants measured in the absence of substrates is not rectilinear, while the same plot of the constants measured in the presence of substrates is a straight line. Limited proteolysis experiments suggest that aspartate aminotransferase from S. solfataricus undergoes a conformational transition induced by the binding of substrates. Another conformational transition which depends on temperature and occurs in the absence of substrates could explain the non-linear Arrhenius plot of the proteolytic kinetic constants. The latter conformational transition might also be related to the functioning of the archaebacterial aminotransferase since the Arrhenius plot of kcat is non-linear as well.
利用有限蛋白酶解对从嗜热古菌嗜热栖热菌（Sulfolobus solfataricus）中分离出的嗜热天冬氨酸转氨酶进行了构象转变分析，并与猪胞质天冬氨酸转氨酶进行了比较。猪胞质天冬氨酸转氨酶是一种经过充分研究的嗜温转氨酶，与该古菌蛋白有大约15%的相似性。嗜热栖热菌的天冬氨酸转氨酶在28位氨基酸残基处被嗜热菌蛋白酶切割，在32和33位氨基酸残基处被胰蛋白酶切割；类似地，猪心脏胞质天冬氨酸转氨酶在19和25位氨基酸残基处被胰蛋白酶切割[伊里亚尔特，A.，于贝尔，E.，克拉夫特，K. & 马丁内斯 - 卡里翁，M.（1984年）《生物化学杂志》259卷，723 - 728页]。就嗜热栖热菌的天冬氨酸转氨酶而言，蛋白酶解切割也会导致转氨酶失活，这表明这两种酶虽然在进化上不同，但都拥有一个参与催化且易被蛋白酶作用的区域，该区域在它们的一级结构中位置相似。据报道，底物的结合会诱导天冬氨酸转氨酶发生构象转变，并保护酶不被蛋白酶解[格林，H.（1985年）《转氨酶》（克里斯滕，P. & 梅茨勒，D. E.编）第323 - 326页，约翰威利父子出版公司，纽约]。嗜热栖热菌的天冬氨酸转氨酶受到底物的保护而不被蛋白酶解，但仅在高温（高于60摄氏度）下。为了解释这种行为，在25 - 75摄氏度的温度范围内测量了嗜热菌蛋白酶导致的失活动力学。在无底物情况下测得的蛋白酶解动力学常数的阿累尼乌斯图不是直线，而在有底物情况下测得的常数的同一图是一条直线。有限蛋白酶解实验表明，嗜热栖热菌的天冬氨酸转氨酶会发生由底物结合诱导的构象转变。另一种依赖温度且在无底物时发生的构象转变可以解释蛋白酶解动力学常数的非线性阿累尼乌斯图。后一种构象转变也可能与古菌转氨酶的功能有关，因为催化常数（kcat）的阿累尼乌斯图也是非线性的。
