Chymotrypsin isoenzymes in Atlantic cod; differences in kinetics and substrate specificity

Area of Science:

• Biochemistry
• Enzymology
• Comparative Proteomics

Background:

• Chymotrypsin isoenzymes play crucial roles in protein digestion.
• Understanding variations in fish digestive enzymes is important for aquaculture and comparative biology.
• Cod (Gadus morhua) possess unique digestive enzyme systems adapted to their diet.

Purpose of the Study:

• To characterize and compare the kinetic properties and substrate specificities of two cod chymotrypsin isoenzymes, ChT1 and ChT2.
• To elucidate the structural differences between ChT1 and ChT2 through N-terminal amino acid sequencing.
• To compare cod chymotrypsin isoenzymes with those found in higher animals.

Main Methods:

• Enzymatic assays using chromogenic peptide and proteinaceous substrates to determine kinetic parameters (kcat, KM).
• Cleavage site specificity analysis using oxidised B-chain of bovine insulin.
• Direct N-terminal amino acid sequence analysis of the purified isoenzymes.

Main Results:

• ChT1 and ChT2 exhibited different kinetics, with ChT1 showing significantly lower KM values than ChT2.
• ChT1 displayed broader cleavage site specificity compared to ChT2 when using insulin B-chain.
• N-terminal sequencing revealed that both active forms comprised two polypeptide chains (A and B), with variations in the A-chain sequences between ChT1 and ChT2, and compared to bovine chymotrypsin.

Conclusions:

• Cod chymotrypsin isoenzymes ChT1 and ChT2 possess distinct biochemical characteristics, suggesting specialized roles in digestion.
• The observed differences in substrate specificity and kinetics are comparable to those between chymotrypsin C and A/B isotypes in higher vertebrates.
• Structural analysis of the N-terminal regions provides insights into the evolutionary divergence of chymotrypsin isoenzymes.