Derst C, Wehner A, Specht V, Röhm K H
Institut für Physiologische Chemie, Philipps-Universität, Marburg, Germany.
Eur J Biochem. 1994 Sep 1;224(2):533-40. doi: 10.1111/j.1432-1033.1994.00533.x.
The importance of five tyrosine residues of Escherichia coli asparaginase II (EcA2) for catalysis and protein stability was examined by site-directed mutagenesis, chemical modification of wild-type and variant enzymes, and by thermodynamic studies of protein denaturation. While the tyrosine residue Y25 is directly involved in catalysis, the hydroxyl groups of residues Y181, Y250, Y289 and Y326 are not necessary for EcA2 activity. However, residues Y181 and Y326 are crucial for stabilization of the native EcA2 tetramer. pH titration curves showed that the active-site residue Y25 has a normal pKa while the C-terminal Y326 is unusually acidic. 1H-NMR signals of a peculiar ligand-sensitive tyrosine residue were assigned to Y25. These and other data suggest that a peptide loop (residues 14-27) which shields the active site during catalysis is highly flexible in the free enzyme.
通过定点诱变、野生型和变体酶的化学修饰以及蛋白质变性的热力学研究，考察了大肠杆菌天冬酰胺酶II（EcA2）的五个酪氨酸残基对催化作用和蛋白质稳定性的重要性。虽然酪氨酸残基Y25直接参与催化作用，但残基Y181、Y250、Y289和Y326的羟基对于EcA2活性并非必需。然而，残基Y181和Y326对于天然EcA2四聚体的稳定至关重要。pH滴定曲线表明，活性位点残基Y25具有正常的pKa，而C端的Y326酸性异常。一个特殊的对配体敏感的酪氨酸残基的1H-NMR信号被确定为Y25。这些数据以及其他数据表明，在催化过程中屏蔽活性位点的一个肽环（残基14 - 27）在游离酶中具有高度的灵活性。
