A comparison of dogfish and bovine chymotrypsins

Abstract

Bovine and dogfish chymotrypsins were compared to determine if chymotrypsin from a poikilothermic organism (spiny dogfish ( Squalus acanthias)) adapted to low temperatures possessed catalytic properties different from those of the same enzyme from a warm-blooded animal. An improved procedure was developed for isolating dogfish pancreatic chymotrypsin. The least hydrophobic and smallest substrate used, p-nitrophenyl acetate, had similar enthalpies of association ( ΔH a ) with both enzymes, whereas larger, more hydrophobic substrates had ΔH a values that were of opposite sign for the two enzymes. As the temperature increased, the association constants ( 1K s ) for p-nitrophenyl valerate and p-nitrophenyltrimethyl acetate increased for dogfish chymotrypsin and decreased for bovine chymotrypsin, while the free energies of association ( ΔG a ) remained relatively constant. Acylation of chymotrypsin was 1.5–2.5 times slower in the dogfish enzyme than in the bovine enzyme except below 15 °C with p-nitrophenyltrimethyl acetate. ΔH‡ for acylation by p-nitrophenyltrimethyl acetate were 2.0 kcal/ mol for the dogfish enzyme and 10.2 kcal/mol for the bovine, whereas ΔH‡ values were only slightly lower in the dogfish enzyme for the other two substrates. For all substrates, the deacylation rate constant ( k cat ) was greater with dogfish chymotrypsin than bovine. However, the free energies of activation ( ΔG‡) for deacylation were nearly equal between the two enzymes for each of the substrates.