Hydrolysis of beta-casein by gastric proteases. I. Comparison of proteolytic action of bovine chymosin and pepsin A

Hydrolysis of beta A2-casein by bovine chymosin and pepsin A was performed in order to compare the hydrolysis of the two enzymes on this protein. Different conditions have been tested: pH 5.5 for 116h and pH 3.5 for 7 h [E/S = 1/100 (w/w)] for chymosin. pH 3.0 for 24 h [E/S = 1/1000 (w/w)] for pepsin A. Under these conditions 17 peptides were obtained after the action of chymosin and 23 after the action of pepsin A. They corresponded respectively to the cleavage of 14 and 15 peptide bonds for chymosin and pepsin A. However, six of the peptide bonds were only hydrolyzed by chymosin and seven other bonds only by pepsin A. Our results showed a preferential splitting at the Leu-X, Ser-X, and Trp-X bonds for chymosin and Leu-X, Met-X, and Thr-X, for pepsin A. Some of the identified peptides contained sequences with possible physiological roles.     

Kinetic studies on the mechanism of pepsin action.

The linear noncompetitive inhibition of the pepsin-catalyzed hydrolysis of Ac-Phe-Phe-Gly at pH 2.1 by L-Ac-Phe, L-Ac-Phe-NH2, and L-Ac-Phe-OEt has been claimed to substantiate the ordered release of products specified by the amino-enzyme mechanism for pepsin action. According to this interpretation, the binding of inhibitor to free enzyme and the amino-enzyme intermediate (Scheme I) generates the observed inhibition pattern. The proposition is valid only if a simple alternative explanation for the kinetic data, Scheme II, can be disproved. Scheme II attributes the inhibition pattern to the binding of inhibitor to free enzyme and the enzyme-substrate (Michaelis) complex. The experiments reported here have enabled us to distinguish between the two mechanisms. The pepsin-catalyzed hydrolyses of Ac-Phe-Trp, Z-H'IS-Phe-Trp, Z-Gly-His-Phe-Trp, and Z-Ala-His-Phe-Trp at pH 1.8 occur exclusively at the Phe-Trp bond and must yield the same amino-enzyme, E-Trp, if it is implicated. Under these circumstances, Scheme I requires that a plot of 1/kc vs. (I)o for the four substrates and a given noncompetitive inhibitor provide a set of four parallel lines. Scheme II predicts that the four lines generally will not be parallel. L-Ac-Phe, L-Ac-Phe-NH2, L-Ac-Phe-OMe, and D-Ac-Phe act as linear noncompetitive inhibitors for the pepsin-catalyzed hydrolysis of the four Trp-containing substrates. The plot of 1/kc vs. (I)o for each inhibitor results in a set of four nonparallel lines. Therefore Scheme II must be correct and the detection of noncompetitive inhibition accompanying the pepsin-catalyzed hydrolysis of peptides offers no insight into the merits of the amino-enzyme hypothesis.     

Liberation of tryptic fragments from caseinomacropeptide of bovine kappa-casein involved in platelet function. Kinetic study.

Carbohydrate-free caseinomacropeptide (CMP) was purified from rennet-hydrolysed caseinate by trichloroacetic acid precipitation and DEAE-TSK Fractogel-650 ion-exchange chromatography. To study the liberation of 106-112, 106-116 and 113-116 fragments from carbohydrate-free CMP involved in platelet function, a quantitative study was made on the rate of hydrolysis of the three peptidic bonds that are susceptible to the action of trypsin. Data were obtained from reverse-phase (Ultrabase column) and cationic-exchange (Mono S column) h.p.l.c. On the basis of the disappearance of substrate, kcat. and Km were respectively 3.95 s-1 and 0.2 mM. The two 111-112 and 112-113 bonds were split according to similar kinetic parameters (kcat. = 1.97 s-1, Km = 0.2 mM) and much faster than the 116-117 bond. The difference in susceptibility of the bonds can probably be attributed to the nature of residues flanking the primary proteolytic sites rather than to their accessibility to the proteinase. On the basis of our results the 106-116 fragment cannot be formed.     

Role of S'1 loop residues in the substrate specificities of pepsin A and chymosin

Proteolytic specificities of human pepsin A and monkey chymosin were investigated with a variety of oligopeptides as substrates. Human pepsin A had a strict preference for hydrophobic/aromatic residues at P'1, while monkey chymosin showed a diversified preferences accommodating charged residues as well as hydrophobic/aromatic ones. A comparison of residues forming the S'1 subsite between mammalian pepsins A and chymosins demonstrated the presence of conservative residues including Tyr(189), Ile(213), and Ile(300) and group-specific residues in the 289-299 loop region near the C terminus. The group-specific residues consisted of hydrophobic residues in pepsin A (Met(289), Leu/Ile/Val(291), and Leu(298)) and charged or polar residues in chymosins (Asp/Glu(289) and Gln/His/Lys(298)). Because the residues in the loop appeared to be involved in the unique specificities of respective types of enzymes, site-directed mutagenesis was undertaken to replace pepsin-A-specific residues by chymosin-specific ones and vice versa. A yeast expression vector for glutathione-S-transferase fusion protein was newly developed for expression of mutant proteins. The specificities of pepsin-A mutants could be successfully altered to the chymosin-like preference and those of chymosin mutants, to pepsin-like specificities, confirming residues in the S'1 loop to be essential for unique proteolytic properties of the enzymes. An increase in preference for charged residues at P'1 in pepsin-A mutants might have been due to an increase in the hydrogen-bonding interactions. In chymosin mutants, the reverse is possible. The changes in the catalytic efficiency for peptides having charged residues at P'1 were dominated by k(cat) rather than K(m) values.     

Kinetics of action of chymosin (rennin) on some peptide bonds of bovine beta-casein

The first steps of proteolysis of bovine beta-casein by chymosin were studied quantitatively by using reverse-phase high-performance liquid chromatography (RP-HPLC). Although chymosin has a broad specificity, it has been possible to selectively study the hydrolysis of two bonds (Ala-189-Phe-190 and Leu-192-Tyr-193) by choosing appropriate conditions. The disappearance of the substrate and the appearance of the reaction products as a function of time were followed at 220 nm by RP-HPLC. For concentrations where beta-casein was in a micellar form, the Michaelian parameters corresponding to the cleavage of bond 192-193 were determined by measuring initial rates of reaction at different substrate concentrations in a time period for which splitting of bond 189-190 was negligible. The following results were obtained; k1cat = 1.54 s-1, K1m = 0.075 mM, and k1cat/K1m = 20.6 mM-1 s-1. Under conditions where the protein was in a monomeric state, the following parameters were determined for the splitting of bond 192-193 by integrating the Michaelis equation: k2cat = 0.056 s-1, K2m = 0.007 mM, and k2cat/K2m = 79.7 mM-1 s-1. Under the latter conditions the four enzymic reactions involved in the cleavage of bonds 189-190 and 192-193 were first-order reactions. The four corresponding apparent rate constants were calculated by using a computer program. Excellent agreement was obtained between concentrations of four molecular species measured during the reaction period and those calculated by using the four apparent rate constants.     

Release and recovery of porcine pepsin and bovine chymosin from reverse micelles: a new technique based on isopropyl alcohol addition.

After complete solubilization by the direct method, porcine pepsin was not released from AOT in isooctane reverse micelles even under aqueous-phase conditions which would not ordinarily allow uptake. Similarly, bovine chymosin, once forward-transferred at a pH below its isoelectric point, was not back-transferred into an aqueous contact phase buffered at a pH value above its isoelectric point. These results show that there is significant hysteresis in the forward- and backward-transfer processes and further imply that kinetics, and not equilibrium, control uptake or release processes for these enzymes. The addition of 10-15% isopropyl alcohol to the aqueous phase increases the rate of protein release dramatically and allows for nearly complete back-transfer of porcine pepsin and 70% back-transfer of bovine chymosin. IPA addition does not destroy the functional integrity of the system since forward transfer of bovine chymosin still occurs at pH values below (but not above) the pI of the protein.     

Specificity of chymosin on immobilized bovine B-chain insulin

1. The specificity of chymosin on immobilized bovine B-chain insulin is studied. 2. Eight sites of hydrolysis are determined.     

Identification of human milk kappa-casein on polyacrylamide gels by differential staining with Ethyl-Stains-all and chymosin sensitivity

Ethyl-Stains-all (ESA), a cationic carbocyanine dye that stains phosphorylated, sialylated, and unmodified proteins differentially, was used to stain a human casein fraction enriched for its kappa-casein-like characteristics. The staining properties and chymosin sensitivity of this fraction were compared with those of human milk and bovine casein proteins. Phosphorylated human and bovine beta caseins stained blue with ESA. The sialic acid-containing bovine kappa-casein stained blue-green. The human kappa-like fraction was enriched for a protein that stained blue-green with ESA. Both bovine kappa-casein and the human blue-green-staining protein were susceptible to chymosin digestion at lower concentrations of chymosin than that required for digestion of beta-caseins. In each case, following chymosin digestion, a green-staining peptide of lower molecular weight replaced the original protein and para-kappa-casein was formed. Identification of human kappa-casein on SDS-polyacrylamide gels was based on its differential staining with ESA and chymosin sensitivity with respect to beta-casein.     

Separation of chymosin and pepsin in calf rennet by dye-ligand affinity chromatography

When calf rennet containing approximately 15% pepsin was applied to a Cibacron Blue agarose column at pH 5.5 in a low salt medium, pepsin passed through unadsorbed while chymosin was bound to the gel in the column. After washing the column, the bound chymosin was eluted with 1.7 M NaCl or 50% (v/v) aqueous ethylene glycol. The salt eluate was analyzed and found to contain greater than 97% pure chymosin. The fraction that passed through unadsorbed was found to contain greater than 96% pure pepsin. Thus a complete separation of chymosin and pepsin was effected by this technique without having to destroy either enzyme. Both enzymes are highly negatively charged at pH 5.5 but the separation does not arise from anion exchange since the gel functions as a cation exchanger. The separation appears to result from a combination of hydrophobic and electrostatic interactions of chymosin with Blue agarose. It is suggested that the enhanced affinity of chymosin to the Blue gel over pepsin may arise from topographically specified interaction between chymosin and the blue chromophore. Differential surface hydrophobicity may also play a key role, since in the presence of 0.7 M Na2SO4 the same behavior as at low ionic strength is observed.     

A PCR-RFLP test to detect allelic variants of the bovine kappa-casein gene

Point mutations in exon IV of bovine kappa-casein gene (kappaCn, CASK, CSN3) determine nine allelic variants (A, B, C, E, F, G, H, I, and A1) for the gene. These variants are associated with major differences in composition and manufacturing properties of milk (i.e., cheese yield). A PCR-RFLP test was developed in order to distinguish the different alleles. Polymorphisms are detected by digestion with the endonucleases HindIII, HaeIII, and MaeII followed by electrophoresis in agarose gels stained with ethidium bromide. Twenty eight DNA samples from different breeds of Argentina were analyzed for the A, B, and E variants. This simple PCR-RFLP test makes feasible the inclusion of kappa-casein genotypes in breeding plans.     

The three-dimensional structure of recombinant bovine chymosin at 2.3 A resolution

The crystal structure of recombinant bovine chymosin (EC 3.4.23.4; renin), which was cloned and expressed in Escherichia coli, has been determined using X-ray data extending to 2.3 A resolution. The crystals of the enzyme used in this study belong to the space group I222 with unit cell dimensions alpha = 72.7 A, b = 80.3 A, and c = 114.8 A. The structure was solved by the molecular replacement method and was refined by a restrained least-squares procedure. The crystallographic R factor is 0.165 and the deviation of bond distances from ideality is 0.020 A. The resulting model includes all 323 amino acid residues, as well as 297 water molecules. The enzyme has an irregular shape with approximate maximum dimensions of 40 x 50 x 65 A. The secondary structure consists primarily of parallel and antiparallel beta-strands with a few short alpha-helices. The enzyme can be subdivided into N- and C-terminal domains which are separated by a deep cleft containing the active aspartate residues Asp-34 and Asp-216. The amino acid residues and waters at the active site form an extensive hydrogen-bonded network which maintains the pseudo 2-fold symmetry of the entire structure. A comparison of recombinant chymosin with other acid proteinases reveals the high degree of structural similarity with other members of this family of proteins as well as the subtle differences which make chymosin unique. In particular, Tyr-77 of the flap region of chymosin does not hydrogen bond to Trp-42 but protrudes out in the P1 pocket forming hydrophobic interactions with Phe-119 and Leu-32. This may have important implications concerning the mechanism of substrate binding and substrate specificity.     

Specificity of milk-clotting enzymes towards bovine kappa-casein

Limited proteolysis of bovine kappa-casein has been investigated with porcine pepsin A and C, and with the 2 microbial proteinases Mucor miehei proteinase and Endothia parasitica proteinase. The liberated C-terminal glycomacropeptide of kappa-casein was isolated after precipitation in 3% trichloroacetic acid followed by high-performance gel-permeation chromatography on a TSK G3000 SW column. From amino acid analyses and N-terminal sequencing of the liberated peptide it is concluded that porcine pepsin A, C and Mucor miehei proteinase cleave the same bond as chymosin: Phe-105-Met-106 whereas Endothia parasitica proteinase cleaves the bond Ser-104-Phe-105.     

牛凝乳酶基因在毕赤酵母中的重组表达

通过PCR技术从克隆载体pMD18T-Prochy上扩增牛凝乳酶原基因,双酶切后定向插入到酵母表达载体pPICZaA中,构建表达质粒pPICZaA-Prochy,线性化后电转化毕赤酵母GS115,经PCR和测序鉴定凝乳酶原基因成功插入到毕赤酵母的基因组中。在甲醇诱导下进行凝乳酶的表达,SDS-PAGE分析证明重组凝乳酶的分子量约为37 kD,培养基上清液中凝乳酶的活性为12.2 SU/mL。本研究首次应用毕赤酵母表达牛凝乳酶,在培养基中获得分泌表达的重组凝乳酶,为干酪工业提供了新型及优良的凝乳酶来源。     

The multimeric structure and disulfide-bonding pattern of bovine kappa-casein.

Bovine kappa-casein was analyzed by SDS/PAGE, MS and amino acid sequence analysis in order to determine its multimeric composition and disulfide-bonding pattern. SDS/PAGE revealed that kappa-casein in the native state can range in size from a monomer to a multimeric structure larger than a decamer. Three types of interchain disulfide linkage, Cys11-Cys11, Cys11-Cys88 and Cys88-Cys88, were all assigned in multimers purified from [14C]carboxymethylated and untreated bulk milk, as well as a milk sample from a kappa-casein-variant-B homozygote Co20. These results indicate that multimerization occurs in a random or at present unpredictable disulfide-bonding pattern regardless of the size of the multimer or the genotype.