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Primary structure of Streptomyces griseus metalloendopeptidase II
S Kojima1, T Kumazaki, S Ishii
1Institute for Biomolecular Science, Gakushuin University, Tokyo, Japan.
Abstract:
Streptomyces griseus metalloendopeptidase II (SGMPII) is a unique protease, since it shows anomalous susceptibility to the proteinaceous "serine protease inhibitors" produced by Streptomyces, such as Streptomyces subtilisin inhibitor (SSI) and its homologous proteins. In this study, we analyzed the amino acid sequence of SGMPII by analyzing various peptide fragments produced enzymatically. The sequence of SGMPII, which is composed of 334 amino acids, showed no extensive similarity to SSI-insensitive metalloproteases produced by other species of Streptomyces, except for the amino acid residues essential for catalysis and zinc binding. However, SGMPII is 35-41% similar to thermolysin and its related metalloproteases, which are not inhibited by SSI, and the residues presumed to be critical for catalysis and zinc-binding are well conserved in SGMPII. Glu137 in a "His-Glu-Xaa-His" motif of SGMPII was identified as the residue modified by CICH2 CO-DL-(N-OH)Leu-Ala-Gly-NH2, an active-site-directed irreversible inhibitor of thermolysin-like metalloproteases. Based on the sequence comparison of SGMPII and other bacterial metalloproteases, we discuss the structural basis for the differences in substrate specificity and stability between SGMPII and other thermolysin-like proteases. A possible SSI-binding locus of SGMPII is also proposed.
Insights
Streptomyces griseus metalloendopeptidase II (SGMPII) is a unique protease inhibited by serine protease inhibitors. Sequence analysis reveals conserved catalytic residues, suggesting structural insights into its unusual inhibition.
Area of Science:
- Biochemistry
- Enzymology
- Protease research
Background:
- Streptomyces griseus metalloendopeptidase II (SGMPII) exhibits unusual susceptibility to Streptomyces-derived serine protease inhibitors (SSI).
- Understanding the structural basis for this unique inhibitory interaction is crucial for protease research.
Purpose of the Study:
- To analyze the amino acid sequence of SGMPII.
- To investigate the structural basis for SGMPII's unique susceptibility to SSI.
- To compare SGMPII with other metalloproteases.
Main Methods:
- Enzymatic fragmentation and peptide analysis to determine SGMPII's amino acid sequence.
- Sequence comparison with known metalloproteases, including thermolysin.
- Active-site-directed irreversible inhibition using a thermolysin-like metalloprotease inhibitor.
Main Results:
- SGMPII (334 amino acids) shows limited similarity to SSI-insensitive metalloproteases but conserves catalytic and zinc-binding residues.
- SGMPII shares 35-41% similarity with thermolysin and related SSI-insensitive metalloproteases.
- Glutamate 137 (Glu137) within the 'His-Glu-Xaa-His' motif was identified as the inhibited residue.
Conclusions:
- SGMPII's unique inhibition by SSI is likely due to structural differences compared to other thermolysin-like proteases.
- Conserved catalytic residues suggest SGMPII belongs to the thermolysin-like metalloprotease family.
- A potential SSI-binding site on SGMPII is proposed, warranting further structural investigation.