Electrophoretic study of the caseinolytic activity of a pepsin from the dogfish Scyliorhinus canicula

Electrophoretic patterns of casein and casein subfractions were studied following proteolysis by dogfish pepsin II or calf chymosin. Both enzymes hydrolyze the kappa casein subfraction with the production of kappa paracasein peptide. alpha S1 and beta subfractions hydrolysis is stronger with dogfish enzyme than with chymosin. It is concluded that, despite a broader specificity, the activity spectrum of dogfish enzyme is, in many respects, similar to that of calf chymosin.     

Kinetic study of the action of bovine chymosin and pepsin A on bovine kappa-casein

A study was carried out to determine the Michaelian parameters relative to the action of chymosin and pepsin A on bond Phe105-Met106 of bovine kappa0-casein (carbohydrate-free fraction in micellar state). The reaction was performed in citrate buffer, pH 6.2, at 30 degrees C. The reaction mixture was analysed by reverse phase HPLC. Dosages of peptide 106-169 (caseino macropeptide) at different reaction times from recordings of its absorbance at 220 nm gave the initial rates of reaction at each substrate concentration. From these values the following parameters were determined: kcat = 68.5 s-1, Km = 0.048 mM, kcat/Km = 1,413 mM-1 s-1 for chymosin, and kcat = 45 s-1, Km = 0.018 mM, kcat/Km = 2,439 mM-1 s-1 for pepsin A. For chymosin they are similar to those obtained previously in dimethyl glutarate buffer, pH 6.6, at 30 degrees C, using fragment 98-111 of kappa-casein as substrate. It can thus be concluded that neither the micellar state nor the presence of the whole peptide chain of kappa-casein (our conditions) significantly affect the action of chymosin on fragment 98-111, which seems to contain all information that makes bond 105-106 highly sensitive to chymosin. For pepsin A, only the information contained in fragment 103-108 appears to be required.     

Kinetic studies on the action of Mucor pusillus, Mucor miehei acid proteases and chymosins A and B on a synthetic chromophoric hexapeptide

The action of two milk-clotting fungal proteases from Mucos pusillus and Mucor miehei and of chymosins A and B on the hexapeptide, Leu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe, and on kappa-casein were studied. The effects of pH and temperature on the initial rates of hydrolysis of the hexapeptide were examined. Crystalline chymosin and M. pusillus protease exhibited optimal activities around 49 and 55 degrees C, respectively, whereas the optimum temperature for M. miehei protease is higher than 63 degrees C. The optimum pH was about 4.7 for both fungal proteases whereas chymosin A and chymosin B exhibited optimal activities around 4.2 and 3.7, respectively. Kinetic parameters were then determined under optimal conditions and/or at pH 4.7. Fungal proteases had kcat/Km ratios that were similar to each other and that were significantly greater than the ratios obtained for the chymosins. Nevertheless, chymosins had much greater clotting activities towards kappa-casein relative to their proteolytic activities towards the synthetic peptide.     

Characterization of bovine kappa-casein fractions and the kinetics of chymosin-induced macropeptide release from carbohydrate-free and carbohydrate-containing fractions determined by high-performance gel-permeation chromatography.

Bovine kappa-casein was fractionated at pH 8.0 on DEAE-Sepharose with an NaCl gradient, followed by DEAE-cellulose chromatography using a decreasing pH gradient from pH 6.0 to 4.5. At least ten components could be identified, each differing in N-acetylneuraminic acid (NeuAc) and/or phosphorus content. Two components appeared to be multiply-phosphorylated, but did not contain NeuAc. The possible significance of this finding in relation to the mode of phosphorylation and glycosylation in vivo is discussed. A carbohydrate-free fraction as well as two NeuAc-containing fractions were compared in their substrate behaviour towards the action of the milk-clotting enzyme chymosin at pH 6.6 and 30 degrees C. To this end the trichloroacetic acid-soluble reaction products were analysed by high-performance gel-permeation chromatography. In order of increasing carbohydrate content the kcat. values found ranged from 40 to 25 s-1 and the Km values from 9 to 3 microM; the overall substrate properties of these components as reflected by the kinetic parameter kcat./Km ranged from 5 to 8 microM-1 X S-1. Irreversible polymerization of the carbohydrate-free fraction brought about a more-than-2-fold increase in Km, the kcat. value remaining virtually constant. The kcat./Km found for the cleavage of whole kappa-casein at pH 6.6 was of the same magnitude as the kcat./Km found for the polymerized carbohydrate-free fraction (i.e. about 3 microM-1 X S-1). No indication of substrate inhibition was found for the carbohydrate-free fraction.     

Isolation and partial characterization of prochymosin and chymosin from cat

Extracts of cat gastric mucosa contain a zymogen that after activation shows partial immunochemical identity with chymosin (EC 3.4.23.4) from calf. Cat prochymosin has been purified by column chromatography and gel filtration, and cat chymosin was obtained after acid activation of the zymogen. The enzyme showed the optimum of general proteolytic activity at pH 2.5. The amino acid compositions of cat prochymosin and chymosin were similar to those of the corresponding proteins from calf. The first 27 residues of both cat prochymosin and chymosin have been sequenced. Among these 54 positions only 13 differences have been observed between the proteins from cat and calf. The results support the hypothesis that the chymosins form a group of neonatal gastric proteases with high milk-clotting activity, but with such weak general proteolytic activity that postnatal uptake of IgG is not hindered.     

Purification and characterization of carboxypeptidase from terminally differentiated rat epidermal cells

A tissue carboxypeptidase-A-like enzyme was purified to apparent homogeneity from terminally differentiated epidermal cells of 2-day-old rats by potato inhibitor affinity chromatography followed by FPLC Mono Q column chromatography. The enzyme has an Mr of 35,000 as determined by SDS-polyacrylamide gel electrophoresis and HPLC gel filtration. It has a pH optimum of 8.5 for hydrolysis of benzyloxycarbonyl-Phe-Leu (Km = 0.22 mM, kcat = 57.9 s-1). The enzyme does not hydrolyze substrates with Arg, Lys and Pro at the C-terminal and Pro at the penultimate position. Angiotensin I was effectively hydrolyzed (Km = 0.06 mM, kcat = 6.48 s-1) and produced both des-Leu10-angiotensin I and angiotensin II. The enzyme activity, relatively stable at 4 degrees C and pH 8.0-10.5, was inactivated at pH values higher than 12.0 and lower than 5.0 or at 65 degrees C for 10 min. Inhibitor profiles of the epidermal enzyme also differed slightly from those of tissue carboxypeptidase A of pancreatic or mast cell origin.     

Comparison of vertebrate collagenase and gelatinase using a new fluorogenic substrate peptide

A fluorogenic substrate for vertebrate collagenase and gelatinase, Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2, was designed using structure-activity data obtained from studies with synthetic inhibitors and other peptide substrates of collagenase. Tryptophan fluorescence was efficiently quenched by the NH2-terminal dinitrophenyl group, presumably through resonance energy transfer. Increased fluorescence accompanied hydrolysis of the peptide by collagenase or gelatinase purified from culture medium of porcine synovial membranes or alkali-treated rabbit corneas. Amino acid analysis of the two product peptides showed that collagenase and gelatinase cleaved at the Gly-Leu bond. The peptide was an efficient substrate for both enzymes, with kcat/Km values of 5.4 microM-1 h-1 and 440 microM-1 h-1 (37 degrees C, pH 7.7) for collagenase and gelatinase, respectively. Under the same conditions, collagenase gave kcat/Km of about 46 microM-1 h-1 for type I collagen from calf skin. Since both enzymes exhibited similar Km values for the synthetic substrate (3 and 7 microM, respectively), the higher catalytic efficiency of gelatinase reflects predominantly an increase in kcat. Both enzymes were inhibited by HSCH2(R,S)CH[CH2CH(CH3)2]CO-L-Phe-L-Ala-NH2 in this assay (50% inhibition at 20 nM and less than 1 nM for collagenase and gelatinase, respectively). Soluble type I collagen was a competitive inhibitor of peptide hydrolysis by collagenase (KI = 0.8 microM) and exhibited mixed inhibition of gelatinase (KI = 0.3 microM).     

Kinetics of the action of chymosin (rennin) on a peptide bond of bovine alpha s1-casein. Comparison of the behaviour of this substrate with that of beta- and kappa o-caseins

The action of chymosin on the Phe23-Phe24 bond of bovine alpha s1-casein, in citrate buffer (pH 6.2) at 30 degrees C, was followed by reversed-phase HPLC quantification of residual alpha s1-casein or fragment 24-199 after different time periods and at different substrate concentrations. This allowed determination of the Michaelian parameters for the reaction under study which were compared with those previously obtained for the action of chymosin on beta- and kappa o-casein under identical reaction conditions. The whole efficiency of the three reactions, as estimated by kcat/Km, was 1.8, 20.6 and 1405.0 for alpha s1-, beta- and kappa o-caseins, respectively. The specificity of chymosin is discussed in the light of these results and of the known sequences of the 3 caseins.     

Isolation of Milk-Clotting Enzyme from Transgenic Sheep Milk and Its Comparison with Calf Chymosin

Technology for preparation of chymosin from milk of transgenic sheep has been elaborated.Purification of the preparation by ion-exchange chromatography on aminosilochrom and biospecific chromatography on bacitracin-Sepharose yielded homogeneous active enzyme. Hydrolysis of protein substrates (hemoglobin, BSA, and sodium caseinate) by the transgenic sheep chymosin and stability of the enzyme at various values of pH were studied. Judging by the amino acid composition, the N-terminal sequence involving six amino acid residues, molecular mass, stability at various pH values, and the cat alytic activity against the protein substrates, the transgenic sheep chymosin is identical to calf chymosin.     

Alteration of aspartate 101 in the active site of Escherichia coli alkaline phosphatase enhances the catalytic activity

The function of aspartic acid residue 101 in the active site of Escherichia coli alkaline phosphatase was investigated by site-specific mutagenesis. A mutant version of alkaline phosphatase was constructed with alanine in place of aspartic acid at position 101. When kinetic measurements are carried out in the presence of a phosphate acceptor, 1.0 M Tris, pH 8.0, both the kcat and the Km for the mutant enzyme increase by approximately 2-fold, resulting in almost no change in the kcat/Km ratio. Under conditions of no external phosphate acceptor and pH 8.0, both the kcat and the Km for the mutant enzyme decrease by approximately 2-fold, again resulting in almost no change in the kcat/Km ratio. The kcat for the hydrolysis of 4-methyl-umbelliferyl phosphate and p-nitrophenyl phosphate are nearly identical for both the wild-type and mutant enzymes, as is the Ki for inorganic phosphate. The replacement of aspartic acid 101 by alanine does have a significant effect on the activity of the enzyme as a function of pH, especially in the presence of a phosphate acceptor. At pH 9.4 the mutant enzyme exhibits 3-fold higher activity than the wild-type. The mutant enzyme also exhibits a substantial decrease in thermal stability: it is half inactivated by treatment at 49 degrees C for 15 min compared to 71 degrees C for the wild-type enzyme. The data reported here suggest that this amino acid substitution alters the rates of steps after the formation of the phospho-enzyme intermediate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of protons on the amidase activity of human alpha-thrombin. Analysis in terms of a general linkage scheme

The amidase activity of human alpha-thrombin has been studied in the pH range 5.5 to 10, and at four different chloride concentrations from 5 mM to 1 M. The Michaelis-Menten constant, Km, shows a bell-shaped dependence over the pH range studied, with a minimum around pH 8. The pH dependence of the catalytic constant, kcat, shows multiple inflection points especially at low (less than 0.1 M) chloride concentrations, thereby implicating the existence of multiple catalytic forms of the enzyme. A general linkage scheme is proposed for the analysis of the effect of protons on thrombin amidase activity, and experimental data have globally been analysed over the entire pH range in terms of such a scheme. Four proton-linked ionizable groups seem to be involved in the control of thrombin amidase activity. Two of these groups change their pK value upon substrate binding to the enzyme and account for the pH dependence of Km. All four groups control the catalytic activity of the enzyme which decreases with increasing protonation. Chloride has little effect on Km, while kcat changes significantly at pH less than 8. This effect is due to an increased enzymatic activity of the highly protonated intermediates at high chloride concentrations, as well as to the pK shift of two proton-linked ionizable groups.     

Kinetic studies of wheat carboxypeptidase-catalyzed reaction: differences in pressure and temperature dependence of peptidase and esterase activities

A kinetic study of hydrolytic catalysis by wheat bran carboxypeptidase (carboxypeptidase W) was carried out using 3-(2-furyl)acryloyl-acylated (Fua-) synthetic substrates. This enzyme showed high esterase activity in addition to the intrinsic carboxypeptidase activity. The optimum pH for the peptidase activity (kcat/Km) was at pH 3.3 and the kcat/Km value decreased with increasing pH with an apparent pKa of 4.50, while the esterase activity increased with pH up to pH 8 with an apparent pKa of 6.04. Optimum pH's for kcat for the peptidase and esterase reactions were also very different and their apparent pKa values were 3.80 and 6.15, respectively. From a measurement of the pressure dependences of kcat and Km, the activation volumes (delta V not equal to) and reaction volumes (delta V), respectively, were determined. delta V not equal to for kcat was -7 to -8 ml/mol for peptidase and -2 to -3 ml/mol for esterase. These results lead us to propose that the peptidase and esterase activities of carboxypeptidase W are different not in the rate-determining steps in a common reaction pathway, but in the binding modes and/or catalytic site(s).     

Kinetic study of o-dianisidine oxidation by hydrogen peroxide in the presence of horseradish peroxidase]

A kinetic study of o-dianisidine oxidation by hydrogen peroxide in the presence of horseradish peroxidase within the pH range of 3.7-9.0 has been carried out. It was shown that the reaction of o-dianisidine peroxidase oxidation obeys the Michaelis--Menten kinetics; the kcat and Km values within the pH range used were determined. The optimum of peroxidase catalytic activity during o-dianisidine oxidation was observed at pH 5.0-6.0. The kinetic pattern of the reaction is discussed. It was demonstrated that deprotonation of the group at pK 6.5 decreases the kcat value 60 times. At pH greater than 8.0 an additional ionogenic group controls the enzyme activity.     

Secondary enzyme-substrate interactions: kinetic evidence for ionic interactions between substrate side chains and the pepsin active site

The possibility that pig pepsin has a cation binding specificity in its secondary binding subsites has been examined by the pepsin-catalyzed hydrolysis of a series of synthetic octa- to undecapeptide substrates. These chromophoric substrates are cleaved by pepsin in the phenylalanyl-p-nitrophenylalanyl (Phe-Nph) bond. Lys and Arg residues were placed into seven different positions in the substrates, and their effect on kcat and Km was examined between pH 2.8 and pH 5.8 (I = 0.1 M, 37 degrees C). Kinetic evidence indicates the existence in the enzyme binding subsites S4, S3, S2, S3', S4', and S5' of a group(s) which become(s) negatively charged at higher pH. For most substrates, the magnitude as well as the pH dependence of kcat was unaffected by the presence of Lys or Arg in these peptides. In contrast, changes up to 5 orders of magnitude were observed for Km, depending on the number of basic residues and on their positions in the sequence. Km for a group of substrates at pH greater than 5.5 was lower than 50 nM. Values for kcat/Km for some substrates exceed the level of 10(8) M-1 s-1. Therefore, the free energy derived from ionic interactions in secondary binding sites influences mostly the binding step on the reaction pathway. This result is in contrast to the previous observations that the length and the hydrophobic character of the substrate residues in some positions influence kcat with little effect on Km toward shorter substrates of pepsin [Fruton, J. (1976) Adv. Enzymol. Relat. Areas Mol. Biol. 44, 1-36].     

The pH dependence of the hydrolysis of chromogenic substrates of the type, Lys-Pro-Xaa-Yaa-Phe-(NO2)Phe-Arg-Leu, by selected aspartic proteinases: evidence for specific interactions in subsites S3 and S2

Variation in the kinetic parameters, kcat and Km, with pH has been used to obtain evidence for significant acid-dissociation processes in the hydrolysis of octapeptide substrates by three aspartic proteinases. These substrates are all cleaved at the peptide bond between a Phe (P1) and a p-nitroPhe (P1') residue resulting in a shift in absorbance at 300 nm that facilitates kinetic measurements. The substrates differ in the amino-acid residues present in the P3 and the P2 positions. Porcine pepsin, calf chymosin, and the aspartic proteinase from Endothia parasitica all show pH dependencies that imply that favorable or unfavorable interactions can occur with the S3 or S2 areas of the enzyme-active site. Examination of the crystallographically determined structure of the E. parasitica proteinase and consideration of the amino-acid sequence differences between the three enzymes suggests that the origin of the pH effects arises from favorable interactions between Glu-13 (COO-) of pig pepsin and Thr (OH) or His (ImH+) in P3 of a substrate. Similarly, Lys-220 (NH3+) of chymosin and a Glu (COO-) in P2 of a substrate may produce a favorable interaction and Asp-77 (COO-) of E. parasitica proteinase and a Glu (COO-) in P2 of a substrate may produce an unfavorable interaction. These results lead to possible explanations for subtle specificity differences within a family of homologous enzymes, and suggest loci for study by site-directed mutagenesis.     

Dependence of the P2-S2 stereochemical selectivity of papain on the nature of the catalytic-site chemistry. Quantification of selectivity in the catalysed hydrolysis of the enantiomeric N-acetylphenylalanylglycine 4-nitroanilides.

1. N-Acetyl-L-phenylalanylglycine 4-nitroanilide and its D-enantiomer were synthesized and characterized and used as substrates with which to evaluate stereochemical selectivity in papain (EC 3.4.22.2)-catalysed hydrolysis. 2. Kinetic analysis at pH 6.0, I 0.1, 8.3% (v/v) NN-dimethylformamide and 25 degrees C by using initial-rate data with [S] much less than Km and weighted non-linear regression provided values of kcat./Km for the catalysed hydrolysis of both enantiomers as (kcat./Km)L = 2040 +/- 48 M-1.S-1 and (kcat./Km)D = 5.9 +/- 0.07 M-1.S-1. These data, taken together with individual values of kcat. and Km for the hydrolysis of the L-enantiomer (a) estimated in the present work as kcat. = 3.2 +/- 1.2 S-1 and Km = 1.5 +/- 0.6 mM and (b) reported by Lowe & Yuthavong [(1971) Biochem. J. 124, 107-115] for the reaction at pH 6.0 in 10% (v/v) NN-dimethylformamide and 35 degrees C, as kcat. = 1.3 +/- 0.2 S-1 and Km = 0.88 +/- 0.1 mM, suggest that (kcat./Km)L congruent to 2000 M-1.S-1 and thus that (kcat./Km)L/(kcat./Km)D congruent to 330.3. Model building indicates that both enantiomeric 4-nitroanilides can bind to papain such that the phenyl ring of the N-acetylphenylalanyl group makes hydrophobic contacts in the S2 subsite with preservation of mechanistically relevant hydrogen-bonding interactions and that the main difference is in the positioning of the beta-methylene group. 4. The dependence of P2-S2 stereochemical selectivity of papain on the nature of the catalytic-site chemistry for reactions involving derivatives of N-acetylphenylalanine is discussed. The variation in the index of stereochemical selectivity (ratio of the appropriate kinetic or thermodynamic parameter for a given pair of enantiomeric ligands), from 330 for the overall acylation process of the catalytic act, through 40 and 31 for the reaction at electrophilic sulphur in 2-pyridyl disulphides respectively without and with assistance by (His-159)-Im(+)-H, to 5 for the formation of thiohemiacetal adducts by reaction at aldehydic carbon, is interpreted in terms of the extent to which conformational variation of the bound ligand in the catalytic-site region permits the binding mode of the -CH2-Ph group of the D-enantiomer to approach that of the L-enantiomer.     

The mechanism of the quinone reductase reaction of pig heart lipoamide dehydrogenase.

The relationship between the NADH:lipoamide reductase and NADH:quinone reductase reactions of pig heart lipoamide dehydrogenase (EC 1.6.4.3) was investigated. At pH 7.0 the catalytic constant of the quinone reductase reaction (kcat.) is 70 s-1 and the rate constant of the active-centre reduction by NADH (kcat./Km) is 9.2 x 10(5) M-1.s-1. These constants are almost an order lower than those for the lipoamide reductase reaction. The maximal quinone reductase activity is observed at pH 6.0-5.5. The use of [4(S)-2H]NADH as substrate decreases kcat./Km for the lipoamide reductase reaction and both kcat. and kcat./Km for the quinone reductase reaction. The kcat./Km values for quinones in this case are decreased 1.85-3.0-fold. NAD+ is a more effective inhibitor in the quinone reductase reaction than in the lipoamide reductase reaction. The pattern of inhibition reflects the shift of the reaction equilibrium. Various forms of the four-electron-reduced enzyme are believed to reduce quinones. Simple and 'hybrid ping-pong' mechanisms of this reaction are discussed. The logarithms of kcat./Km for quinones are hyperbolically dependent on their single-electron reduction potentials (E1(7]. A three-step mechanism for a mixed one-electron and two-electron reduction of quinones by lipoamide dehydrogenase is proposed.     

Kinetic study of carboxypeptidase P-catalyzed reaction. Pressure and temperature dependence of kinetic parameters

Detailed kinetic analyses of carboxypeptidase P-catalyzed reactions were carried out spectrophotometrically using 3-(2-furyl)acryloyl-acylated peptide substrates. The maximum kcat/Km was observed at around pH 3.5 for the synthetic peptide substrates. The kcat/Km value decreased with increasing pH, with an apparent pKa value of 4.43. However, the maximum kcat was observed at neutral pH (pH congruent to 6) and the pKa was 4.49. These apparently different pH profiles for kcat/Km and kcat of this enzyme were due to the decreasing Km value in the acid pH region. The pressure and temperature dependences of these kinetic parameters were also measured. N-Benzoylglycyl-L-phenyllactate (Bz-Gly-OPhLac) gave dependences similar to those of the peptide substrate, suggesting that there is no distinct difference in the catalytic mechanism between the peptide and the ester hydrolyses.     

Demonstration of chymosin (EC 3.4.23.4) in the stomach of newborn pig.

The stomach of newborn pig contains a proteinase that is immunologically closely related to calf chymosin (rennin) (EC 3.4.23.4.). None of the pepsins from the stomach of adult pig is present in the newborn pig. Pig chymosin has optimal general proteolytic activity around pH 3.5. The ratio of milk-clotting activity to general proteolytic activity is about 30--70 times higher than that of pyloric and fundic pepsins.     

Prostaglandin H synthase kinetics. The effect of substituted phenols on cyclooxygenase activity and the substituent effect on phenolic peroxidatic activity.

A series of p- and m-substituted phenols were examined for their effect on the cyclooxygenase activity of prostaglandin H synthase in 0.1 M phosphate buffer at pH 8.0 and 25.0 +/- 0.1 degrees C. A biphasic response was observed. At low concentrations phenols stimulate, but at higher concentrations inhibit, cyclooxygenase activity. Both enhancement and inhibition are increased by phenolic substituents which are electron-donating, quantified by Hammett sigma constants, and hydrophobic, quantified by Hantsch tau constants. The same series of substituted phenols was also reacted with compound II of prostaglandin H synthase at 4.0 +/- 0.5 degrees C. The compound II data fit the Hammett rho sigma equation; no hydrophobicity factors are required. Phenols inhibit cyclooxygenase activity by interfering with the binding of arachidonic acid to compound I and by competing directly with arachidonic acid as reducing substrates for compound I. Phenols stimulate cyclooxygenase activity by acting as reducing substrates for compound II, thereby accelerating the peroxidatic cycle. Phenols also protect the enzyme from self-catalyzed inactivation, most likely by removing the free radical of prostaglandin G2 by reducing it to prostaglandin G2. Kinetic parameters Km and kcat for cyclooxygenase activity were determined in the presence of phenols. Identical values of Km (15.3 +/- 0.5 mM) and kcat (89 +/- 2 s-1) were obtained regardless of which phenol was employed. Therefore these represent the true Km and kcat values for cyclooxygenase activity.