Chymotrypsin titration

Such an intermediate is known to be formed in reactions catalyzed by trypsin, chymotrypsin, thrombin, other enzymes of the blood-clotting cascade (except angiotensinconverting enzyme, which is an aspartic protease). An acyl-serine intermediate is also formed in the acetylcholinesterase reaction. The active site serine of this enzyme and the serine proteases can be alkylated by diisopropyl-fluorophosphate. See also Active Site Titration... 

The resonances of protons in hydrogen bonds may be shifted downfield to such an extent that they may be observed in H20 solutions. The proton between Asp-102 and His-57 in chymotrypsinogen, chymotrypsin, and other serine proteases has been located and its resonance found to titrate with a pKa of 7.518 (although the pKa is for the dissociation of the proton on the other nitnjjgen of the imidazole ring). ... 

For a-chymotrypsin, the procedure of active-site titration for the calculation of active enzyme concentration and thus of the catalytic constant kcat is long established. The original active-site titration experiment on a-CT by Hartley and Kilby (Hartley, 1954) was performed with ethyl p-nitrobenzoate (Figure 9.2). 

Titration of the intact active site obviates problems due to inactive protein which contribute to a false molarity. Active-site titrations of acyl group transfer enzymes such as a-chymotrypsin utilise a substrate which has a good leaving group. This enables the buildup of an acyl enzyme intermediate which forms faster than it can degrade and results in... 

Fig. 11.10 (A) Burst kinetics for release of the leaving group from a substrate of a-chymotrypsin. (B) Titration of papain with the irreversible inhibitor 4-toluenesulphonamidomethyl chloromethyl ketone using methyl benzoylglycinate as substrate.

For Questions 4.14-4.17, use the following information. You have isolated an unknown peptide whose titration curve is presented in Figure 4.16. The peptide absorbed ultraviolet light at 280 nm. Treatment with trypsin released free alanine and arginine. Treatment with chymotrypsin resulted in quantitative production of a neutral tripeptide and an alkaline dipeptide. 

Havsteen and Hess (1962) have studied the titration of the phenolic groups of a-chymotrypsin and of its diisopropylphosphoryl (DIP) derivative. The result is similar to that observed with chymotrypsinogen (Fig. 4) in that only two of the four groups are available for titration in the native protein, as well as in the DIP derivative. The data do not have sufficient precision to determine whether the two titratable groups have different pK s, as they do in chymotrypsinogen, but the wide spread of the titration curve suggests that they do. 

All four tyrosyl groups are titrated normally in solvents which denature chymotrypsin. 

An exchangeable proton of ribonuclease A titrates with a pK of 5.8 and has been assigned [39] to the NH of the active-site histidine-119. A low field resonance can be observed for chymotrypsin in H2O [40] and its pH dependence (15 to 18 ppm, pKa 7.2) and response to chemical modification suggests that this is the hydrogen-bonded proton between His-57 and Asp-102 at the active site. 

Stock solutions of DFP can be conveniently prepared in isopropanol in concentrations from 0.1 M to 0.001 M. These solutions are stable for a month in the refrigerator (Jansen et al. 1949). Moon et al. (1965), in their studies of the reaction of chymotrypsin with DFP, have determined the normality of stock solution of DFP in the following manner. Roughly 0.03 moles of DFP were added to an aqueous solution of 0.17 moles of KOH in a volumetric flask. The solution was allowed to stand for 12 hr at 25°C to permit complete hydrolysis, and was titrated to pH 7.0 with 0.1 N HCl. Identical titrimetric results were obtained after 43 hr standing in alkali indicating that complete hydrolysis had taken place after 12 hr. To determine the amount of free acid, if any, in the DFP, the same amount of DFP that was used... 

N-(m/is-Cinnamoyllmidazole (1). Mol. wt. 198.22, m.p. 133-133.5°. Prepared in high yield by reaction of cinnamoyl chloride with imidazole in benzene at 10-25°. The reagent reacts rapidly and quantitatively with the active site of a-chymotrypsin and hence can be used for the spectrophotometric determination of the normality of an enzyme solution by titration. 

Presented in Fig. 8 are the data on the determination of the minimal amount of water required for a-chymotrypsin catalysis. The system CTAB-dimethyl sulfoxide/water-octane/chloroform was used as a reaction medium, and the volume ratio of water to dimethyl sulfoxide was varied from 0 to 0.001 [12,44]. It can be seen that in the totally dry system the reaction does not take place. Introduction of water activates the enzyme. Its full activation (defined by titration) occurs in the presence of just a few molecules of water (around 5) per enzyme molecule. The plateau in Fig. 8 is explained by the fact that the enzyme used is a hydrolase in low-water conditions acylation of the enzyme by the substrate occurs with quantitative formation of the acyl-enzyme. (Incidentally, it is a direct demonstration of acyl-enzyme formation in the reaction of chymotrypsin with an anilide substrate.) Hydrolysis of acyl-enzyme by water occurs at higher water concentrations, when, apparently, water appears as a reagent in a free state. 

In all cases the addition of the chymotrypsin reversible inhibitor, indole, caused an upfield shift of the native line, i.e. in the direction of the denatured enzyme towards the bulk solution environment. Since we found that all of these spectra met fast chemical exchange conditions an indole titration was possible, as shown in Figure 2, where we have shown the upfield shift VS total indole concentration and the theoretical hyperbolic fit to these data for the o-CF labeled enzyme at pH 4.2. The dissociation constants for the three derivatives were 5.2, 3.2, and 3.7 mM for the o-CF f and p-CF labeled chymotrypsins, respective-... 

Streptomyces griseus trypsin [8] and a-lytic protease [9] are serine proteases with a solitary histidine residue. This histidine residue is believed to be involved in the catalytic mechanism via a charge-relay mechanism analogous to that in a-chymotrypsin. Figures 2 and 3 show the reactivity data for the histidine residue in a-lytic protease and S.G. Trypsin. The data for a-lytic protease approximately follow a titration curve... 

Two general procedures for monitoring the reaction of aza-peptide nitrophenyl esters with serine proteases will be discussed. The first (Ac-Ala-Aphe-ONp -j- chymotrypsin) illustrates the use of enzymes that are readily accessible to the investigator. It is also a situation in which the acylated enzyme has a long lifetime. The second (Ac-Ala-Ala-Aala-ONp elastase) is designed so that only minimal amounts of enzyme need be employed and requires the use of a spectrophotometer with an expanded scale of 0.01 A. In this case, turnover of the acylated enzyme is fairly rapid, but the procedure is useful for titrating elastase. 

Chitin has been used as a matrix for the covalent immobilization of enzymes for example, jS-o-galactosidases, chymotrypsin, and acid phosphatase were attached to chitin via glutaraldehyde. Pretreatment of chitin with acid, base, ammonia, or pronase was compared with regard to the release of titratable amino-groups and the ability of the polysaccharide to retain /S-D-galactosidase activity. 

J. L. Markley and I. B. Ibanez (1978), Zymogen activation in serine proteinases. Proton magnetic resonance pH titration studies of the two histidines of bovine chymotrypsinogen A and chymotrypsin Aa. Biochemistry 17, 4627-4639. 

The importance of accurately determining pKa values of titratable sites of proteins is supported by the large number of studies pertaining to their calculation. In addition to method development " " " and review articles,some of the systems most recently studied are noted here a-chymotrypsin, bacteriorhodopsin, myoglobin, ribonuclease ribonuclease protein BPTI, lyso-... 

Fig. 7. Summary of P-NMR titration data of diisopropylphosphoryl derivatives of serine proteinases. The arrows represent the extent of titration shuts and the numbers indicate pA , values. Abbreviations DIP, diisopropylphosphoryl BCtg, bovine chymotrypsinogen A BTg, bovine trypsinogen PTg, porcine trypsinogen BCtr , bovine chymotrypsin A BTr, bovine trypsin PTr, porcine trypsin a-LP, a-lytic proteinase. From Porcubcu et al. (1979). Copyright 1979 American Chemical Society.

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