Veltman O R, Eijsink V G, Vriend G, de Kreij A, Venema G, Van den Burg B
Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands.
Biochemistry. 1998 Apr 14;37(15):5305-11. doi: 10.1021/bi972374j.
The active site of thermolysin-like proteases (TLPs) is located at the bottom of a cleft between the N- and C-terminal domains. Crystallographic studies have shown that the active-site cleft is more closed in ligand-binding TLPs than in ligand-free TLPs. Accordingly, it has been proposed that TLPs undergo a hinge-bending motion during catalysis resulting in "closure" and "opening" of the active-site cleft. Two hinge regions have been proposed. One is located around a conserved glycine 78; the second involves residues 135 and 136. The importance of conserved glycine residues in these hinge regions was studied experimentally by analyzing the effects of Gly --> Ala mutations on catalytic activity. Eight such mutations were made in the TLP of Bacillus stearothermophilus (TLP-ste) and their effects on activity toward casein and various peptide substrates were determined. Only the Gly78Ala, Gly136Ala, and Gly135Ala + Gly136Ala mutants decreased catalytic activity significantly. These mutants displayed a reduction in kcat/Km for 3-(2-furylacryloyl)-L-glycyl-L-leucine amide of 73%, 62%, and 96%, respectively. Comparisons of effects on kcat/Km for various substrates with effects on the Ki for phosphoramidon suggested that the mutation at position 78 primarily had an effect on substrate binding, whereas the mutations at positions 135 and 136 primarily influence kcat. The apparent importance of conserved glycine residues in proposed hinge-bending regions for TLP activity supports the idea that hinge-bending is an essential part of catalysis.
嗜热菌蛋白酶样蛋白酶（TLPs）的活性位点位于N端和C端结构域之间裂隙的底部。晶体学研究表明，与无配体的TLPs相比，结合配体的TLPs中活性位点裂隙更封闭。因此，有人提出TLPs在催化过程中会发生铰链弯曲运动，导致活性位点裂隙的“闭合”和“打开”。已提出两个铰链区。一个位于保守的甘氨酸78附近；第二个涉及残基135和136。通过分析甘氨酸突变为丙氨酸对催化活性的影响，对这些铰链区中保守甘氨酸残基的重要性进行了实验研究。在嗜热脂肪芽孢杆菌的TLP（TLP-ste）中进行了8个这样的突变，并测定了它们对酪蛋白和各种肽底物活性的影响。只有Gly78Ala、Gly136Ala和Gly135Ala + Gly136Ala突变体显著降低了催化活性。这些突变体对3-（2-呋喃丙烯酰基）-L-甘氨酰-L-亮氨酸酰胺的kcat/Km分别降低了73%、62%和96%。比较各种底物对kcat/Km的影响与对磷酰胺脒Ki的影响表明，78位的突变主要影响底物结合，而135和136位的突变主要影响kcat。在提出的铰链弯曲区域中保守甘氨酸残基对TLP活性的明显重要性支持了铰链弯曲是催化重要组成部分的观点。
