Probing Catalytic Hinge Bending Motions in Thermolysin-like Proteases by Glycine Alanine Mutations
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- 作者：Oene R. Veltman ; Vincent G. H. Eijsink ; Gert Vriend ; Arno de Kreij ; Gerard Venema ; and Bertus Van den Burg
- 刊名：Biochemistry
- 出版年：1998
- 出版时间：April 14, 1998
- 年：1998
- 卷：37
- 期：15
- 页码：5305 - 5311
- 全文大小：323K
- 年卷期：v.37,no.15(April 14, 1998)
- ISSN：1520-4995
文摘
The active site of thermolysin-like proteases (TLPs) is located atthe bottom of a cleft betweenthe N- and C-terminal domains. Crystallographic studies have shownthat the active-site cleft is moreclosed in ligand-binding TLPs than in ligand-free TLPs.Accordingly, it has been proposed that TLPsundergo a hinge-bending motion during catalysis resulting in"closure" and "opening" of the active-sitecleft. Two hinge regions have been proposed. One is locatedaround a conserved glycine 78; the secondinvolves residues 135 and 136. The importance of conserved glycineresidues in these hinge regions wasstudied experimentally by analyzing the effects of Gly 
Alamutations on catalytic activity. Eight suchmutations were made in the TLP of Bacillusstearothermophilus (TLP-ste) and their effects onactivitytoward casein and various peptide substrates were determined. Onlythe Gly78Ala, Gly136Ala, andGly135Ala + Gly136Ala mutants decreased catalytic activitysignificantly. These mutants displayed areduction in kcat/Km for3-(2-furylacryloyl)-L-glycyl-L-leucine amide of73%, 62%, and 96%, respectively.Comparisons of effects onkcat/Km for varioussubstrates with effects on the Ki forphosphoramidon suggestedthat the mutation at position 78 primarily had an effect on substratebinding, whereas the mutations atpositions 135 and 136 primarily influence kcat.The apparent importance of conserved glycine residuesinproposed hinge-bending regions for TLP activity supports the idea thathinge-bending is an essential partof catalysis.
Alamutations on catalytic activity. Eight suchmutations were made in the TLP of Bacillusstearothermophilus (TLP-ste) and their effects onactivitytoward casein and various peptide substrates were determined. Onlythe Gly78Ala, Gly136Ala, andGly135Ala + Gly136Ala mutants decreased catalytic activitysignificantly. These mutants displayed areduction in kcat/Km for3-(2-furylacryloyl)-L-glycyl-L-leucine amide of73%, 62%, and 96%, respectively.Comparisons of effects onkcat/Km for varioussubstrates with effects on the Ki forphosphoramidon suggestedthat the mutation at position 78 primarily had an effect on substratebinding, whereas the mutations atpositions 135 and 136 primarily influence kcat.The apparent importance of conserved glycine residuesinproposed hinge-bending regions for TLP activity supports the idea thathinge-bending is an essential partof catalysis.