Subtilisin modification of monodeamidated ribonuclease-A

Area of Science:

• Biochemistry
• Enzymology
• Protein Chemistry

Background:

• Ribonuclease A (RNAase-A) is a well-studied enzyme whose structure-function relationship is critical in molecular biology.
• Limited proteolysis is a key mechanism for enzyme activation and modification, as exemplified by the generation of RNAase-S from RNAase-A.
• Understanding modifications to RNAase-A, such as deamidation, is important for studying enzyme stability and function.

Purpose of the Study:

• To investigate the limited proteolysis of monodeamidated ribonuclease-A (RNAase-Aa(1)) by subtilisin.
• To characterize the resulting RNAase-S type derivative (RNAase-Aa(1)S) and compare its properties to native RNAase-S.
• To explore the structural and functional consequences of deamidation on ribonuclease activity and modification.

Main Methods:

• Limited proteolysis of RNAase-Aa(1) using subtilisin.
• Chromatographic separation and purification of the resulting enzyme derivatives.
• Fractionation of the derivative into protein and peptide components using trichloroacetic acid.
• Enzymic activity assays.
• Susceptibility studies using trypsin.
• Titration experiments to assess protein-peptide binding.
• Acid treatment studies on RNAase-S and RNAase-A.

Main Results:

• Subtilisin digestion of RNAase-Aa(1) produced an active RNAase-S derivative, RNAase-Aa(1)S, with distinct chromatographic properties from RNAase-S.
• RNAase-Aa(1)S exhibited similar enzymic activity, antigenic conformation, and trypsin susceptibility to RNAase-S.
• Fractionation yielded inactive RNAase-Aa(1)S-protein and RNAase-Aa(1)S-peptide, which could regenerate active enzyme upon mixing.
• RNAase-Aa(1)S-peptide was identical to RNAase-S-peptide, but RNAase-Aa(1)S-protein differed from RNAase-S-protein.
• Deamidated protein showed weaker binding to S-peptide compared to native S-protein.
• Subtilisin digestion of RNAase-Aa(1) resulted in only 50% conversion, attributed to product inhibition.
• Acid treatment of RNAase-S affected both protein and peptide regions, unlike RNAase-A.

Conclusions:

• Monodeamidation of RNAase-A alters the protein component, leading to a distinct RNAase-S derivative with comparable, yet subtly different, biochemical properties.
• The observed weaker binding of S-peptide to the deamidated protein suggests conformational changes affecting the active site.
• Product inhibition limits the efficiency of subtilisin digestion for the deamidated substrate.
• Acid lability differs between RNAase-A and RNAase-S, with the latter showing broader susceptibility to acid-induced structural changes.