The primary structure and enzymic properties of porcine prochymosin and 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 comparison of their structures may therefore contribute to an understanding of the significance of the amino acid residues responsible for the differences in these properties. Pig chymosis was extracted from the stomachs of pigs of less than 3 weeks of age, and was purified by ion exchange chromatography. Half of the primary structure was determined by amino acid sequencing and the complete structure was deduced from a cloned chymosin cDNA. Results showed that the zymogen showed 81% sequence identity with calf prochymosin and 57% identity with pig pepsinogen A. The size of the propart and location of the residue which becomes the N-terminus in the active molecule were the same in the prochymosins. The maximum general proteolytic activity at pH 3.5 of pig chymosin was 2-3% of that of the activity of pig pepsin A at pH 2, whereas the milk clotting activity relative to the general proteolytic activity of pig chymosin was much higher than that of calf chymosin. Agar gel electrophoresis at pH 5.3 of stomach extracts of individual pigs showed the 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 properties of the genetic variants were observed. It was concluded that of the residues that participate in the substrate binding, calf and pig chymosin differ in the following positions (pig pepsin numbering, subsites in parentheses): Ser 12 Thr (S4), Leu 30 Val (S1/S3), His 74 Gln (S'2), Val 111 Ile (S1/S3), Lys 220 Met (S4). With regard to the low general proteolytic activity of pig chymosin, the substitution Asp 303 Val relative to calf chymosin may contribute to an explanation of this.
Houen
G
G
Department of Autoimmunology, Statens Seruminstitut, Copenhagen, Denmark.
Madsen
M T
MT
Harlow
K W
KW
Lønblad
P
P
Foltmann
B
B
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001483:Base Sequence; D012085:Chymosin / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D004792:Enzyme Precursors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D013329:Structure-Activity Relationship; D013552:Swine
D000596:Amino Acids; D004792:Enzyme Precursors; C032443:prorennin; D012085:Chymosin