Probing catalytic hinge bending motions in thermolysin-like proteases by glycine --> alanine mutations

Biochemistry
O R VeltmanBertus van den Burg

Abstract

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 a...Continue Reading

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Citations

Dec 26, 2002·Journal of Molecular Recognition : JMR·Günther H Peters, Robert P Bywater
Jun 8, 2011·Protein Science : a Publication of the Protein Society·V G H EijsinkG Vriend
Jan 20, 2011·Proteins·Sebastian Radestock, Holger Gohlke
Feb 23, 2002·The Journal of Biological Chemistry·Arno de KreijJens E Nielsen
May 16, 2002·The Journal of Biological Chemistry·Hiroki Tsukada, Tayebeh Pourmotabbed
Nov 21, 2001·The Journal of Biological Chemistry·Daan M F van AaltenWim Crielaard

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