THE PRIMARY STRUCTURE AND ENZYMATIC-PROPERTIES OF PORCINE PROCHYMOSINAND CHYMOSIN

Preliminary investigations by N-terminal sequence analysis showed that pig and calf chymosin possessed 80% amino acid sequence identity but showed considerable differences in their enzymatic properties. A compa rison of their structures may therefore contribute to an understanding of the significance of the amino acid residues responsible for the di fferences in these properties. Pig chymosis was extracted from the sto machs of pigs of less than 3 weeks of age, and was purified by ion exc hange chromatography. Half of the primary structure was determined by amino acid sequencing and the complete structure was deduced from a cl oned chymosin cDNA. Results showed that the zymogen showed 81% sequenc e identity with calf prochymosin and 57% identity with pig pepsinogen A. The size of the propart and location of the residue which becomes t he N-terminus in the active molecule were the same in the prochymosins . The maximum general proteolytic activity at pH 3.5 of pig chymosin w as 2-3% of that of the activity of pig pepsin A at pH 2, whereas the m ilk clotting activity relative to the general proteolytic activity of pig chymosin was much higher than that of calf chymosin. Agar gel elec trophoresis at pH 5.3 of stomach extracts of individual pigs showed th e existence of two predominant genetic variants of zymogen and enzyme. The two variants could not be distinguished by amino acid composition or N-terminal sequencing, and no differences in the enzymatic propert ies of the genetic variants were observed. It was concluded that of th e residues that participate in the substrate binding, calf and pig chy mosin differ in the following positions (pig pepsin numbering, subsite s in parentheses): Ser 12 Thr (S-4), Leu 30 Val (S-1/S-3), His 74 Gln (S'(2)), Val 111 Re (S-1/S-3), Lys 220 Met (S-4). With regard to the l ow general proteolytic activity of pig chymosin, the substitution Asp 303 Val relative to calf chymosin may contribute to an explanation of this. (C) 1996 Elsevier Science Ltd