Determination of three amino acids causing alteration of proteolytic activities of staphylococcal glutamyl endopeptidases.
Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus warneri secrete glutamyl endopeptidases, designated GluV8, GluSE, and GluSW, respectively. The order of their protease activities is GluSE < GluSW << GluV8. In the present study, we investigated the mechanism that causes these differences. Expression of chimeric proteins between GluV8 and GluSE revealed that the difference is primarily attributed to amino acid residues 170-195, which define the intrinsic protease activity, and additionally to residues 119-169, which affect the proteolytic sensitivity. Among nine substitutions present in residues 170-195 of the three proteases, the substitutions at positions 185, 188, and 189 were responsible for the changes in their activities, and the combination of W185, V188, and P189, which naturally occurs in GluV8, exerts the highest protease activity. W185 and P189 were indispensable for full activity, but V188 could be replaced by hydrophobic amino acids. These three amino acid residues appear to create a substrate-binding pocket together with the catalytic triad and the N-terminal V1, and therefore define the K(m) values of the proteases. We also describe a method to produce a chimeric form of GluSE and GluV8 that is resistant to proteolysis, and therefore possesses 4-fold higher activity than the wild-type recombinant GluV8.
Nemoto
Takayuki K
TK
Department of Oral Molecular Biology, Course of Medical and Dental Sciences, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8588, Japan. tnemoto@nagasaki-u.ac.jp
Ono
Toshio
T
Shimoyama
Yu
Y
Kimura
Shigenobu
S
Ohara-Nemoto
Yuko
Y
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D006868:Hydrolysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D013210:Staphylococcus / Q000201:enzymology*
D000596:Amino Acids; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase