Identification and Designing of the S3 Site of Aqualysin I, a Thermophilic Subtilisin-Related Serine Protease

Aqualysin I is a bacterial subtilisin-related alkaline serine protease, originating in Thermits aquaticus YT-1. Based on computational analysis, we predicted that two residues, Ser102 and Gly131, form the S3 site of aqualysin I, and we proved that this prediction by site-directed mutagenesis. To alt...

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Bibliographic Details
Published in:Journal of biochemistry (Tokyo) 1999-06, Vol.125 (6), p.1016-1021
Main Authors: Tanaka, Terumichi, Matsuzawa, Hiroshi, Ohta, Takahisa
Format: Article
Language:English
Subjects:
Amino Acid Sequence
aqualysin I
Base Sequence
Catalytic Domain - genetics
DNA Primers - genetics
Drug Design
Kinetics
Models, Molecular
Mutagenesis, Site-Directed
Oligopeptides
P3-specificity
Protein Conformation
S3 subsite
Serine Endopeptidases - chemistry
Serine Endopeptidases - genetics
Serine Endopeptidases - metabolism
serine protease
Substrate Specificity
Subtilisins - chemistry
Subtilisins - genetics
Subtilisins - metabolism
subtilsin
Thermus - enzymology
Thermus - genetics
Thermus aquaticus
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Summary:Aqualysin I is a bacterial subtilisin-related alkaline serine protease, originating in Thermits aquaticus YT-1. Based on computational analysis, we predicted that two residues, Ser102 and Gly131, form the S3 site of aqualysin I, and we proved that this prediction by site-directed mutagenesis. To alter the P3-specificity of the enzyme, we built a “wall” on the S3 site edge by introducing a bulky side chain at target sites. Six mutant proteins were prepared: S102H, S102K, S102E, G131H, G131K, and G131D. The mutant enzymes were examined with two kinetically typical peptides for aqualysin I, suc-X-Ala-Ala-pNA, where X is Ala or Phe. All mutations reduced the efficiency for the Phe-containing peptide, while they raised the kcat values for the Ala-containing peptide. Especially, the S102K mutant protein hydrolyzed the polyalanine peptide efficiently. The strategies we have adopted in this paper are applicable to all subtilisin-related enzymes.
ISSN:0021-924X
DOI:10.1093/oxfordjournals.jbchem.a022380