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Avian retroviral protease and cellular aspartic proteases are distinguished by activities on peptide substrates
1Fox Chase Cancer Center, Institute for Cancer Research, Philadelphia, Pennsylvania 19111.
Abstract:
The avian sarcoma/leukemia virus protease (PR), purified from avian myeloblastosis virus has a native molecular mass of 26 kDa, suggesting a dimer structure. The enzymatic activity of PR has been characterized using synthetic peptide substrates. PR is most active at pH 5.5, 35 degrees C and 2-3 M NaCl. Under these conditions PR cleaves decapeptides which are resistant in low ionic strength. This high, nonphysiological, salt concentration also increases the proteolytic activity of a cellular aspartic protease, pepsin. PR and pepsin show additional similarities: they both cleave a synthetic decapeptide at the same Tyr-Pro bond in low and high salt, while the cleavage site preferences of human renin and cathepsin-D in this substrate are altered at high salt concentrations. In addition, iodination of the tyrosine residue in this decapeptide causes an increase in the rates of hydrolysis by both PR and pepsin. However, Km values are too high to be estimated accurately for PR using Tyr-Pro and Tyr(I)-Pro decapeptides as substrates. Comparison of the digestion products of two additional decapeptides, altered in a single amino acid residue, shows that PR cleaves at fewer sites than all three cellular enzymes. Furthermore pepstatin, a strong inhibitor of pepsin, renin, and cathepsin-D has little effect on PR.
Insights
Avian sarcoma/leukemia virus protease (PR) functions optimally in high salt conditions, differing from cellular proteases. This viral protease exhibits unique substrate cleavage patterns and is unaffected by pepstatin, a common inhibitor.
Area of Science:
- Biochemistry
- Enzymology
- Virology
Background:
- The avian sarcoma/leukemia virus protease (PR) is a viral enzyme crucial for viral replication.
- Understanding its enzymatic properties is key to developing antiviral strategies.
Purpose of the Study:
- To characterize the enzymatic activity and substrate specificity of avian sarcoma/leukemia virus protease (PR).
- To compare the properties of PR with cellular aspartic proteases.
Main Methods:
- Purification of avian sarcoma/leukemia virus protease (PR).
- Enzymatic assays using synthetic peptide substrates under varying conditions (pH, temperature, salt concentration).
- Comparative analysis of cleavage sites with cellular proteases (pepsin, renin, cathepsin-D).
Main Results:
- PR exhibits optimal activity at pH 5.5, 35°C, and high salt concentrations (2-3 M NaCl).
- PR cleaves decapeptides resistant at low ionic strength, showing altered substrate preferences compared to cellular enzymes at high salt.
- Iodination of tyrosine enhanced hydrolysis rates for PR and pepsin, but PR's Km values were too high for accurate estimation.
- PR cleaved fewer sites than cellular enzymes and was insensitive to pepstatin.
Conclusions:
- Avian sarcoma/leukemia virus protease (PR) possesses unique biochemical properties, including high salt optimum and distinct substrate specificity.
- PR's insensitivity to pepstatin suggests it may represent a novel target for antiviral drug development.