Trypsin hydrolysis studies

The digestion of growth hormones with carboxypep-tidase, trypsin, and chymotrypsin yields undialyzable polypeptides that maintain full activity thus suggesting that the large molecule contains multiple active centers. Such a finding does not simplify the identification of the active center indeed, the product of the chymotrypsin and trypsin hydrolysis splits at least 30% of the peptide bonds to yield a complex population of polypeptides that are difficult to separate. Further chemical studies have established that the tyrosine side chain and the s-amino groups of lysine are required for growth hormone activity. 

Wang, S.S., and Carpenter, F.H. (1968) Kinetic studies at high pH of the trypsin-catalyzed hydrolysis of Na-benzoyl derivatives of L-arginamide, L-lysinamide, and S-2-aminoethyl-L-cysteinamide and related compounds./. Biol. Chem. 243, 3702-3710. 

There is currently little understanding of the influence of interfacial composition and (nano)structure on the kinetics of enzymatic hydrolysis of biopolymers and lipids. However, a few preliminary studies are beginning to emerge (McClements et al., 2008 Dickinson, 2008). Thus, for example, Jourdain et al. (2009) have shown recently that, in a mixed5 sodium caseinate + dextran sulfate system, the measured interfacial viscosity increased from qs = 220 mN s m 1 without enzyme to qs = 950 mN s m 1 with trypsin present. At the same time, the interfacial elasticity was initially slightly reduced from (7S = 1.6 mN m 1 to (h = 0.7 mN m, although it later returned to close to its original value. Conversely, in the... 

The use of enzymes for solubilization of seed and leaf proteins has been studied as a means of overcoming difficulties presented by the varying condition of seed and leaf material available for processing (37). Ultrasonic energy was reported to increase the efficiency of enzyme solubilization procedures. The effects of various conditions of hydrolysis of cottonseed and alfalfa meal protein with trypsin were defined. 

From study of peptides formed by partial hydrolysis of the 32P-labeled chymotrypsin, the sequence of amino acids surrounding the reactive serine was established and serine 195 was identified as the residue whose side chain hydroxyl group became phosphorylated. The same sequence Gly-Asp-Ser-Gly was soon discovered around reactive serine residues in trypsin, thrombin, elastase, and in the trypsin-like cocoonase used by silkmoths to escape from their cocoons.198 We know now that these are only a few of the enzymes in a very large family of serine proteases, most of which have related active site sequences.199 200 Among these are thrombin and other enzymes of the blood-clotting cascade (Fig. 12-17), proteases of lysosomes, and secreted proteases. 

In Table II are shown the results from kinetic studies with commercially available gastric and pancreatic enzymes. Trypsin was strongly inhibited, at least at a low concentration of casein as substrate. The hydrolysis of benzoyl arginine ethyl ester (BAEE) by trypsin was non-competitively inhibited, giving a 30% reduction of Vmax at 0.5 mg/ml of the LMW fraction. Carboxypepti-dase A, and to a lesser extent carboxypeptidase B, were non-competitively inhibited as well. Pepsin and chymotrypsin were not affected by the conditions used in these assays. 

Of special interest is the reaction of an acyl enzyme with active-site-directed inhibitors in comparison with the free enzyme. Therefore, we studied the hydrolysis of the substrate Bz-Arg-pNA by trypsin and benzoyl-trypsin in the presence of the naturally occurring inhibitor aptotinin [21]. The substrate is cleft... 

Enzymatic gelation of partially heat-denatured whey proteins by trypsin, papain, pronase, pepsin, and a preparation of Streptomyces griseus has been studied (Sato et al., 1995). Only peptic hydrolysate did not form a gel. The strength of the gel depended on the enzyme used and increased with increasing DH. Hydrolysis of whey protein concentrate with a glutamic acid specific protease from Bacillus licheniformis at pH 8 and 8% protein concentration has been shown to produce plastein aggregates (Budtz and Nielsen, 1992). The viscosity of the solution increased dramatically during hydrolysis and reached a maximum at 6% DH. Incubation of sodium caseinate with pepsin or papain resulted in a 55-77% reduction in the apparent viscosity (Hooker et al., 1982). 

Synthetic substrates allow rapid determination of the catalytic constants of an enzyme. Nevertheless, it is known that the environment of the peptide bond depends largely on physico-chemical conditions of the applied media, and imposed steric hindrance. Since these parameters are important, the hydrolysis of purified (3-casein was studied at different pHs. The kinetic analysis revealed that the mutant conserved the native trypsin capacity to hydrolyze peptide bonds containing arginyl and lysyl residues. The optimal pH of activity changed considerably according to the mutation. 

Modifications introduced by the mutations were central to the alteration of the specificities of the enzymes studied, which were capable of cleaving (3-casein at many new sites, for example, hydrolyzing the fragment Argl-Lysl05, reported to be a trypsin inhibitor (Bouhallab et al, 1997). Since many tryptic inhibitors contain amidated Glu and Asp, and form amyloid structures, the mutants of this type could be used for the hydrolysis of the lytically resistant protein structures. 

In addition, peptides binding different minerals have been found in whey proteins, i.e., from (3-lg, a-la and LF. Since these proteins are not phosphorylated, the minerals seem to bind through other binding sites than caseins. Seventeen (17) different peptides have been identified by hydrolysis of (3-lg with thermolysin using two different concentrations of calcium. Also, peptides from a-la and LF using trypsin, chymotrypsin or pepsin have been reported. Studies with 3-lg and a-la peptides have shown a higher affinity for iron than the native proteins (Vegarud et al., 2000). 

Plasmin Hydrolysis of /3-Casein. Studies on the susceptibility of partially methylated /3-casein to cleavage by trypsin-like enzymes were carried out using the enzyme porcine plasmin. In preliminary investigations, we confirmed that the y-caseins produced by plasmin hydrolysis of native /3-casein were identical with those occurring naturally (28). These are designated yi-, y2-, and y3-casein according to the nomenclature recommendations of Whitney et al. (29) and correspond to residues 29-109, 106-209, and 198-209 of -casein. Presumably the proteose peptone products of plasmin hydrolysis are identical with their natural counterparts, but the latter were not available for comparison. 

The results on the hydrolysis of partially methylated /3-casein by plasmin indicate that proteins radiomethylated to a low level can serve as substrates for trypsin-like enzymes and probably for proteinases in general. Because it is likely that methylation will interfere with enzymatic attack at lysine residues, the complete hydrolysis of /3-casein probably would not be possible. Studies on mastitic milk demonstrate the usefulness of 14C-methyl proteins for qualitative examination of protein hydrolysis in complex multiprotein systems where resolution and characterization of individual protein fragments is difficult. The requirements in such studies are the availability of pure samples of the proteins under investigation and a suitable technique for separating the radio-labeled protein from hydrolytic products. 

Considerable effort has been applied to studies of ester hydrolysis catalyzed by imidazoles (76MI40700, 80AHC(27)241). Certainly, 1-acetylimidazole can be made enzymically, probably by the sequence acetyl phosphate + coenzyme A acetylcoenzyme A+phosphate, acetyl-coenzyme A + imidazole l-acetylimidazole+coenzyme A. In addition, the imidazolyl group of histidine appears to be implicated in the mode of action of such hydrolytic enzymes as trypsin and chymotrypsin, thereby engendering further interest in the process of imidazole catalysis. The two pathways which have been found to be involved are general base catalysis and nucleophilic catalysis. In the former (Scheme 26) a basic imidazole molecule can activate a water molecule to attack the ester at the carbonyl carbon, this being followed by the usual sequence of steps as in simple hydroxide ion hydrolysis. At high imidazole concentrations the imidazole molecules may be involved directly. 

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