Baird Teaster T, Wright William D, Craik Charles S
University of California, San Francisco, Department of Pharmaceutical Chemistry, San Francisco, California 94143-2280, USA.
Protein Sci. 2006 Jun;15(6):1229-38. doi: 10.1110/ps.062179006. Epub 2006 May 2.
The hydroxyl group of a serine residue at position 195 acts as a nucleophile in the catalytic mechanism of the serine proteases. However, the chemically similar residue, threonine, is rarely used in similar functional context. Our structural modeling suggests that the Ser 195 --> Thr trypsin variant is inactive due to negative steric interaction between the methyl group on the beta-carbon of Thr 195 and the disulfide bridge formed by cysteines 42 and 58. By simultaneously truncating residues 42 and 58 and substituting Ser 195 with threonine, we have successfully converted the classic serine protease trypsin to a functional threonine protease. Substitution of residue 42 with alanine and residue 58 with alanine or valine in the presence of threonine 195 results in trypsin variants that are 10(2) -10(4) -fold less active than wild type in kcat/KM but >10(6)-fold more active than the Ser 195 --> Thr single variant. The substitutions do not alter the substrate specificity of the enzyme in the P1'- P4' positions. Removal of the disulfide bridge decreases the overall thermostability of the enzyme, but it is partially rescued by the presence of threonine at position 195.
丝氨酸蛋白酶催化机制中，195位丝氨酸残基的羟基作为亲核试剂。然而，化学性质相似的苏氨酸残基在类似功能环境中很少被使用。我们的结构模型表明，Ser 195→Thr胰蛋白酶变体无活性，这是由于195位苏氨酸β-碳上的甲基与42位和58位半胱氨酸形成的二硫键之间存在负空间相互作用。通过同时截短42位和58位残基并将195位丝氨酸替换为苏氨酸，我们成功地将经典丝氨酸蛋白酶胰蛋白酶转化为功能性苏氨酸蛋白酶。在195位苏氨酸存在的情况下，将42位残基替换为丙氨酸，58位残基替换为丙氨酸或缬氨酸，得到的胰蛋白酶变体在kcat/KM方面比野生型活性低10²-10⁴倍，但比Ser 195→Thr单变体活性高>10⁶倍。这些替换不会改变酶在P1'-P4'位置的底物特异性。去除二硫键会降低酶的整体热稳定性，但195位苏氨酸的存在可部分挽救这一情况。
