Protein Engineering, Vol 11, 1205-1210, Copyright © 1998 by Oxford University Press
H Takagi, M Yamamoto, I Ohtsu and S Nakamori
We analyzed the role played by the conserved Gly154, a constituent of the
P1 substrate-binding pocket of Bacillus subtilis subtilisin E, in the
catalytic properties of the protease. Using an Escherichia coli expression
system, the termination codon at position 154 in subtilisin E was first
introduced to abolish the catalytic activity through truncation of the
C-terminus from amino acid residues 154-275. We then attempted to obtain
revertants with substitutions of various amino acids at position 154 by the
polymerase chain reaction using a mixture of oligonucleotides. In addition
to the Gly residue (wild-type), six amino acid substitutions (Ala, Arg,
Leu, Phe, Pro and Thr) gave caseinolytic activity. When assayed with
synthetic peptide substrates, most of the revertants showed a considerable
decrease in specific activity and a P1 specificity similar to that of the
wild-type enzyme. An Ala154 mutant purified from the periplasmic space in
E. coli, however, resulted in an up to 2.3-fold preference for Val rather
than Pro as a P2 substrate relative to the wild-type. Further, a
significant 2-10-fold increase in the catalytic efficiency occurred in the
Gly127Ala plus Gly154Ala combination variant, relative to the single
Gly127Ala variant, without any change in the restricted specificity. The
kinetic data and molecular modeling analysis demonstrate the important role
of position 154 in the catalytic efficiency as well as in the substrate
specificity of subtilisin E.
ARTICLES
Random mutagenesis into the conserved Gly154 of subtilisin E: isolation and characterization of the revertant enzymes
Department of Bioscience, Fukui Prefectural University, Japan.
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