New Chromophoric Peptide Substrates for Chymosin (Rennin)

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Abstract
Kinetic studies on the hydrolysis by chymosin (rennin) of the chromophoric hexapeptide substrates: H-Leu-Thr-Phe(NO2)-Nle-Ala-Leu-OMe, H-Leu-Ala-Phe(NO2)-Nle-Ala-Leu-OMe and H-Leu-D-Ser-Phe(NO2)-Nle-Ala-Leu-OMe allow the conclusion that the .beta.-OH group of Ser104 residue of the physiological substrate (k-casein) is involved in the interactions between chymosin and its substrates. To contribute to effectiveness of these interactions the seryl residue has to be in the L-configuration. Kinetic studies of the hydrolysis of penta-, hexa-, hepta- and octapeptide substrates, the structures of which are close to the sequence around the Phe105- Met106 bond of k-casein, show that elongation of the peptide chain produces a significant increase of the rate of hydrolysis of the sensitive bond. The best substrate synthesized is the octapeptide: H-Pro-His-Leu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe. The study of the effect of pH on the kinetic parameters of the hydrolysis of the hexapeptide H-Leu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe shows that the .**GRAPHIC**. ratio is dependent on the ionization of 2 carboxyl groups of the enzyme, the pK of which are 3.3 and 5.7. Comparative study of the hydrolysis of H-Leu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe and H-Leu-Ala-Phe(NO2)-Nle-Ala-Leu-OMe by chymosin, bovine and porcine pepsins also is reported. [This study relates to dairy product preparation].