Evidence for a pH-dependent irreversible formation of a stable conformation of phenacyl-alpha-chymotrypsin.

A reinvestigation of the modification reactions of alpha-chymotrypsin with phenacyl bromide was carried out. Results conclusively demonstrate that the chemically and physically different modified enzymes prepared at pH 4 and at pH 7 both contain the phenacyl group at methionine-192 in the sulphonium salt form. Evidence to suppoort this conclusion derives from 13C nuclear-magnetic-resonance spectroscopic observations on [methylene-13C]phenacyl-enriched enzymes. More conclusively, the methionine-192-containing C-chain, derived by performic acid oxidative cleavage of radioactively-labelled enzyme prepared at pH 7, was shown to contain the phenacyl moiety and to undergo dealkylation by 2-mercaptoethanol with loss of this moiety. In addition, thermolytic cleavage of the high-pH enzyme results in fragmentation of the polypeptide chain in a fashion analogous to model reactions of phenacylmethionyl dipeptides and other methionine-192 sulphonium salts. A rationalization of the unusual nature of the high-pH phenacyl-modified enzyme based on the irreversible formation of stable conformation in which the phenacyl moiety is rigidly located in interior regions of the enzyme is presented and discussed.     

The effects of chemical modification on the refolding transition of alpha-chymotrypsin.

The role of several active site residues of alpha-chymotrypsin in the prototypical refolding transition between active and inactive forms of this enzyme is examined using chemical modification. Oxidation of Met-192 to the sulfoxide results in a derivative which remains entirely in an active state from pH 6 to 9. The derivative becomes inactive only at high pH with pKa = 10.3, delta H0 = 9.5 kcal and delta S0 = -15 eu., indicating the sulfoxide group supplies about 2.1 kcal of active state stabilization relative to the unoxidized methionine side chain. The refolding transition of N-methyl-His-57-alpha-chymotrypsin, in which a nitrogen of the "charge relay" histidine is methylated, displays one ionization process with an apparent pKa of 9.45. The absence of an additional ionization process with a pKa near 7 provides evidence that one of the ionizations in the six state mechanism which describes this transition in alpha-chymotrypsin is linked to the charge relay system. We also demonstrate, using alpha-chymotrypsin, Met-192-sulfoxide-alpha-chymotrypsin and N-methyl-His-57-alpha-chymotrypsin, that the 230 nm circular dichroism band is a quantitative probe of the active-inactive equilibrium, although the chromophore or chromophores responsible for this and another very large negative band at 202 nm have not been identified. Circular dichroism was used to observe the active-inactive equilibrium in methan sulfonyl-alpha-chymotrypsin and phenylmethane sulfonyl-alpha-chymotrypsin. The enhanced stability of the active state of these derivatives relative to alpha-chymotrypsin can be rationalized in terms of steric effects in the substrate side chain binding site.     

On the role of tryptophan residues in the mechanism of action of glyceraldehyde-3phosphate dehydrogenase as tested by specific modification.

A method is described to selectively modify one of the three tryptophan residues of the subunit of glyceraldehyde-3-phosphate dehydrogenase from yeast. As modifying agent dimethyl (2-hydroxy-5-nitrobenzyl) sulfonium bromide was used. The residue which is modified by the procedure described has been identified as Trp-193. There are either one or two molecules of the modifying agent being added to this tryptophan side chain. The modification apparently does not cause a detectable conformational change of the protein as judged from the methods employed. However, the enzymatic activities in the dehydrogenase as well as in the esterase reactions are lost after the modification. It could be established that the modification rendered the enzyme unable to bind the oxidized coenzyme. Also the charge-transfer interaction between enzyme and coenzyme could no longer be observed.     

An essential tryptophan residue for rabbit muscle creatine kinase

The tryptophan residues in rabbit muscle creatine kinase (ATP:creatine N-phosphotransferase, EC 2.7.3.2) have been modified by dimethyl(2-hydroxy-5-nitrobenzyl) sulfonium bromide after reversible protection of the reactive SH groups. The modification of two tryptophan residues as measured by spectrophotometric titration leads to complete loss of enzymatic activity. Control experiments show that reversible protection of the reactive SH groups as S-sulfonates followed by reduction results in nearly quantitative recovery of enzyme activity. The presence of a 410 nm absorption maximum and the decrease in fluorescence of the modified enzyme indicate the modification of tryptophan residues. At the same time, SH determinations after reduction of the modified enzyme show that the reagent has not affected the protected SH groups. Quantitative treatment of the data (Tsou, C.-L. (1962) Sci. Sin. 11, 1535 1558) shows that among the tryptophan residues modified, one is essential for its catalytic activity. The presence of substrates partially protects the modification of tryptophan residues as well as the inactivation, suggesting that the essential tryptophan residue is situated at the active site of this enzyme.     

Myosin light chain kinase binding to plastic

Methionine-81 and/or -8 of the transmembrane sialoglycoprotein, glycophorin A, have been specifically alkylated with ¹³CH₃I to produce the sulfonium ion derivatives [S-[¹³C]methylmethionine-8]glycophorin A and [S-[¹³C]methylmethionine-8 and -81]glycophorin A. ¹³C NMR spectra of these species show that the resonances of the methyl groups of the modified glycophorins occur at 26.1 ppm downfield from Me₄Si. A spin-lattice relaxation time of 0.4 was observed for the ¹³C-enriched methyl resonances of the sulfonium ion derivatives of Met-8 and -81, which corresponds to an effective correlation time of < 2× 10^?10 s. Demethylation of the 2 glycophorin A sulfonium ion species with 2-mercaptoethanol produces native glycophorin A which now has the ε-carbon of the methionine residue(s) 45% isotopically enriched. The ε-carbon of Met-8 was found to occur at 15.7 ppm downfield from Me₄Si whereas the ε-carbon of Met-81 exhibited an unusual chemical shift of 2.0 ppm downfield from Me₄Si. The spin-lattice relaxation time of both resonances was found to be ～0.3 s.     

Structure of 2-hydroxy-5-nitrobenzylated carboxypeptidase A.

Bovine pancreatic carboxypeptidase A (EC 3.4.12.2) was treated with dimethyl (2-hydroxy-5-nitrobenzyl)sulfonium chloride at pH 7.5, resulting in a preparation which consisted primarily of a monohydroxynitrobenzylated derivative of the enzyme. Samples of the hydroxynitrobenzylated enzyme were subjected to tryptic digestion and to cyanogen bromide cleavage, and resulting peptides were isolated chromatographically. One tryptic hydroxynitrobenzyl-containing peptide was isolated; its amino acid composition was that of the N-terminal tryptic segment of carboxypeptidase Agamma (residues 8--35). Likewise, CNBr cleavage of the hydroxynitrobenzylated enzyme revealed that the hydroxynitrobenzyl group resided in the N-terminal fragment, FN (residues 8--22). Neither of these hydroxynitrobenzylated peptides contains Trp, the amino acid residue which is characteristically the site of hydroxynitrobenzylation in proteins, and each was found to contain approximately one less Asx than the corresponding native peptide. Both dansylation and automated Edman degradation procedures revealed that the N-terminal Asn of carboxypeptidase Agamma had been modified by hydroxynitrobenzylation of the enzyme. Thus the sulfonium salt reacts with carboxypeptidase A in the same manner as that established earlier for 2-hydroxy-5-nitrobenzyl bromide (Radhakrishnan, T.M., Bradshaw, R.A., Deranleau, D.A. and Neurath, H. (1970) FEBS Lett. 7, 72--76). Such reactivity of the alpha-amino group presumably reflects its unique location with respect to Trp residues in the tertiary structure of the enzyme.     

Guanosine 5'-O-[S-(3-bromo-2-oxopropyl)]thiophosphate: a new reactive purine nucleotide analog labeling Met-169 and Tyr-262 in bovine liver glutamate dehydrogenase.

A new guanosine nucleotide has been synthesized and characterized: guanosine 5'-O-[S-(3-bromo-2-oxopropyl)]thiophosphate (GMPSBOP), with a reactive functional group which can be placed at a position equivalent to the pyrophosphate region of GTP. This new analog is negatively charged at neutral pH and is similar in size to GTP. GMPSBOP has been shown to react with bovine liver glutamate dehydrogenase with an incorporation of 2 mol of reagent/mol of subunit. The modification reaction desensitizes the enzyme to inhibition by GTP, activation by ADP, and inhibition by high concentrations of NADH, but does not affect the catalytic activity of the enzyme. The rate constant for reaction of GMPSBOP with the enzyme exhibits a nonlinear dependence on reagent concentration with KD = 75 microM. The addition to the reaction mixture of alpha-ketoglutarate, GTP, ADP, or NADH alone results in little decrease in the rate constant, but the combined addition of 5 mM NADH with 0.4 mM GTP or with 10 mM alpha-ketoglutarate reduces the reaction rate approximately 6-fold. GMPSBOP modifies peptides containing Met-169 and Tyr-262, of which Tyr-262 is not critical for the decreased sensitivity of the enzyme toward allosteric ligands. The presence of 0.4 mM GTP plus 5 mM NADH protects the enzyme against reaction at both Met-169 and Tyr-262, but yields enzyme with 1 mol of reagent incorporated/mol of subunit which is modified at an alternate site, Met-469. In the presence of 0.2 mM GTP + 0.1 mM NADH, protection against modification of Tyr-262, but only partial protection against labeling of Met-169, is observed. In contrast, the presence of 10 mM alpha-ketoglutarate + 5 mM NADH protect only against reaction with Met-169. The results suggest that GMPSBOP reacts at the GTP-dependent NADH regulatory site [Lark, R. H., & Colman, R. F. (1986) J. Biol. Chem. 261, 10659-10666] of bovine liver glutamate dehydrogenase, which markedly affects the sensitivity of the enzyme to GTP inhibition. The reaction of GMPSBOP with Met-169 is primarily responsible for the altered allosteric properties of the enzyme.     

Conformational changes and association of chemically modified chymotrypsins

The low pH conformational transitions of a series of modified chymotrypsins have been examined and compared to the association properties of these same proteins in order to determine if the effect of the modification was directly upon the association or indirectly through conformational changes. The modifications of α-chymotrypsin that have been studied are serine-195 modified with acetyl or with phenylmethane sulfonyl groups, His-57 modified by tosylamidophenylethyl chloromethyl ketone or with a methyl group, and Met-192 modified with a nitrophenacyl group. Chymotrypsinogen and native and modified delta-chymotrypsin have also been studied. Ultraviolet difference spectra and optical rotatory dispersion measurements show that the various proteins may be divided into three groups depending upon their response in the low pH transition. These groupings based upon conformational transitions are not divided in the same manner as were those based upon the effect of the modifications on the association process itself. The latter process had earlier been found to correlate with the size of the modification in the active site. From these findings it has been concluded that the effect of the modifications on the association was directly upon the interacting subunit interface and that the observed conformational change is only incidental to this effect. Therefore, the amino acids involved in the association in solution are the same as those involved in the dimerization in the crystal, namely, the active site region of Ser-195, His-57, and Met-192. This is the most direct demonstration that the mode of association of a protein in solution is the same as the mode of association in the crystal.     

alpha-Bromo-4-amino-3-nitroacetophenone, a new reagent for protein modification. Modification of the methionine-290 residue of porcine pepsin.

A new coloured reagent for protein modification, alpha-bromo-4-amino-3-nitroacetophenone (NH2BrNphAc), was synthesized. The reagent was found to alkylate specifically the methionine-290 residue of porcine pepsin below pH 3 at 37 degrees C, which lead to a 45% decrease of enzyme's activity towards haemoglobin. The effect of this reagent as well as that of other phenacyl bromides on the activity of pepsin appeared to be a result of steric hindrance caused by the attachment of bulky reagent residue to the edge of the cleft harbouring the enzyme active site. Only marginal reaction with the co-carboxy group of aspartic acid-315 was found under the above conditions. More pronounced esterification of carboxy groups (up to one residue per enzyme molecule) occurred when the pH was shifted to 5.2. The latter modification had no noticeable effect on enzyme activity, thus disproving a previously held assumption that pepsin inactivation by phenacyl bromide is due to the carboxy-group esterification. alpha-Bromo-4-amino-3-nitroacetophenone forms derivatives with characteristic u.v. spectra when it reacts with methionine, histidine, aspartic and glutamic acid residues, and may be recommended as a reagent for protein modification.     

Reversed micelles of polymeric surfactants in nonpolar organic solvents. A new microheterogeneous medium for enzymatic reactions.

A new microheterogeneous non-aqueous medium for enzymatic reactions, based on reversed micelles of a polymeric surfactant, was suggested. The surfactant termed CEPEI, was synthesized by successive alkylation of poly(ethyleneimine) with cetyl bromide and ethyl bromide and was found to be able to solubilize considerable amounts of water in benzene/n-butanol mixtures. The hydrodynamic radius of polymeric-reversed micelles was estimated to be in the range 22-51 nm, depending on the water content of the system, as determined by means of the quasi-elastic laser-light scattering. Polymeric reversed micelles were capable of solubilizing enzymes (alpha-chymotrypsin and laccase) in nonpolar solvents with retention of catalytic activity. Due to the strong buffering properties of CEPEI over a wide pH range, it could maintain any adjusted pH inside hydrated reversed micelles. It was found that catalytic behavior of enzymes entrapped in polymeric reversed micelles was rather insensitive to the pH of the buffer solution introduced into the system as an aqueous component, but determined mostly by acid-base properties of the polymeric surfactant itself. Both catalytic activity and stability of entrapped alpha-chymotrypsin and laccase were found to increase with increasing water content of the system. Under certain conditions, the entrapment of alpha-chymotrypsin into CEPEI reversed micelles resulted in a considerable increase in catalytic activity and stability as compared to aqueous solution. CEPEI reversed micelles were demonstrated to be promising enzyme carriers for use in membrane reactors. Owing to the large dimensions of CEPEI reversed micelles, they are effectively kept back by a semipermeable membrane, thus allowing an easy separation of the reaction product and convenient recovery of the enzyme.     

Synthesis of AcPheLeuNH(2) by alpha-chymotrypsin in TTAB reversed micelles: Application of response surface methodology to the optimization of the system

The influence of the long chain alcohols, hexanol, octanol, and decanol, as cosurfactants of the reverse micellar system of tetradecyltrimethylammonium bromide on the alpha-chymotrypsin-mediated AcPheLeuNH(2) synthesis was studied. The effect of temperature, buffer molarity, pH, and substrate concentration was also evaluated. The enzyme was chemically modified and the effect of this modification upon the enzyme activity was also analyzed. Octanol allowed a higher activity for both enzyme forms. The peptide synthesis/substrate hydrolysis ratio is independent of the long chain alcohol used. The chemical modification decreases the alpha-chymotrypsin activity under the system conditions studied, but increases the initial velocity of peptide synthesis relative to the ester substrate hydrolysis. The response surface methodology was applied to optimize the dipeptide synthesis in the system containing octanol as cosurfactant. (c) 1994 John Wiley & Sons, Inc.     

Reaction of alkylcobalamins with thiols

Carbon-13 NMR spectroscopy and phosphorus-31 NMR spectroscopy have been used to study the reaction of several alkylcobalamins with 2-mercaptoethanol. At alkaline pH, when the thiol is deprotonated, the alkyl-transfer reactions involve a nucleophilic attack of the thiolate anion on the Co-methylene carbon of the cobalamins, yielding alkyl thioethers and cob(II)alamin. In these nucleophilic displacement reactions cob(I)alamin is presumably formed as an intermediate. The higher alkylcobalamins react more slowly than methylcobalamin. The lower reactivity of ethyl- and propylcobalamin is probably the basis of the inhibition of the corrinoid-dependent methyl-transfer systems by propyl iodide. The transfer of the upper nucleoside ligand of adenosylcobalamin to 2-mercaptoethanol is a very slow process; S-adenosyl-mercaptoethanol and cob(II)alamin are the final products of the reaction. The dealkylation of (carboxymethyl)cobalamin is a much more facile reaction. At alkaline pH S-(carboxymethyl)mercaptoethanol and cob(II)alamin are produced, while at pH values below 8 the carbon-cobalt bond is cleaved reductively to acetate and cob(II)alamin. The reductive cleavage of the carbon-cobalt bond of (carboxymethyl)cobalamin by 2-mercaptoethanol is extremely fast when the cobalamin is in the "base-off" form. Because we have been unable to detect trans coordination of 2-mercaptoethanol, we favor a mechanism that involves a hydride attack on the Co-methylene carbon of (carboxymethyl)cobalamin rather than a trans attack of the thiol on the cobalt atom.     

Raman spectroscopic study of the changes in secondary structure of chymotrypsin: effect of pH and pressure on the salt bridge

Conformational changes of alpha-chymotrypsin, induced by pH and pressure, have been studied with Raman spectroscopy. The secondary structure of alpha-chymotrypsin, chymotrypsinogen and DFP-chymotrypsin has been calculated by a singular value analysis of the Raman amide-I band. The changes in secondary structure, with pH and pressure titration of alpha-chymotrypsin, indicate a conformational transition. The salt bridge between Asp-194 and Ile-16 is disrupted, and the enzyme becomes inactive. No changes are observed for chymotrypsinogen. It is concluded that the proenzyme exhibits the same conformation at different pH values as alpha-chymotrypsin at alkaline pH. The results for DFP-chymotrypsin indicate that the active conformation is stabilized by the presence of the DFP inhibitor in the binding site.     

Enzyme-polyelectrolyte complexes in water-ethanol mixtures: negatively charged groups artificially introduced into alpha-chymotrypsin provide additional activation and stabilization effects

Formation of noncovalent complexes between alpha-chymotrypsin (CT) and a polyelectrolyte, polybrene (PB), has been shown to produce two major effects on enzymatic reactions in binary mixtures of polar organic cosolvents with water. (i) At moderate concentrations of organic cosolvents (10% to 30% v/v), enzymatic activity of CT is higher than in aqueous solutions, and this activation effect is more significant for CT in complex with PB (5- to 7-fold) than for free enzyme (1.5- to 2.5-fold). (ii) The range of cosolvent concentrations that the enzyme tolerates without complete loss of catalytic activity is much broader. For enhancement of enzyme stability in the complex with the polycation, the number of negatively charged groups in the protein has been artificially increased by using chemical modification with pyromellitic and succinic anhydrides. Additional activation effect at moderate concentrations of ethanol and enhanced resistance of the enzyme toward inactivation at high concentrations of the organic solvent have been observed for the modified preparations of CT in the complex with PB as compared with an analogous complex of the native enzyme. Structural changes behind alterations in enzyme activity in water-ethanol mixtures have been studied by the method of circular dichroism (CD). Protein conformation of all CT preparations has not changed significantly up to 30% v/v of ethanol where activation effects in enzymatic catalysis were most pronounced. At higher concentrations of ethanol, structural changes in the protein have been observed for different forms of CT that were well correlated with a decrease in enzymatic activity. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 267-277, 1997.     

Enzymes in polyelectrolyte complexes. The effect of phase transition on thermal stability

Penicillin amidase, alpha-chymotrypsin and urease have been immobilized in water-soluble nonstoichiometric polyelectrolyte complexes (N-PEC). N-PEC are formed by modified poly(N-ethyl-4-vinyl-pyridinium bromide) (polycation) and excess poly(methylacrylic acid) (polyanion). N-PEC are a new class of polymers capable, characteristically, of phase transitions solution in equilibrium precipitate induced by slight change in pH or ionic strength. Neither the chemical structure of the carrier nor the number of cross-linkages between an enzyme and a carrier change on phase transition. That gives an unique opportunity to elucidate the difference between enzymes immobilized on water-soluble and water-insoluble supports. A detailed study of the phase transition effect on thermal stability of the enzymes and protein-protein interactions has been carried out. The following effects were found. Pronounced thermal stabilization of penicillin amidase and urease may be achieved on two conditions: the enzyme is in the precipitate; (b) the enzyme is linked to the N-PEC nucleus. Then the thermal stability of N-PEC-bound penicillin amidase increases 7-fold at pH 5.7, 60 degrees C, and 300-fold at pH 3.1, 25 degrees C, compared to the native enzyme. For urease, the thermal stabilization increases 20-fold at pH 5.0, 70 degrees C. The localization of enzyme on N-PEC has been established by titration of alpha-chymotrypsin bound to a polycation or polyanion with basic pancreatic trypsin inhibitor. Both in solution (pH 6.1) and in N-PEC precipitate (pH 5.7), an alpha-chymotrypsin molecule bound to a polyanion is fully exposed to the solution. If the enzyme is bound to a polycation, only 20% of alpha-chymotrypsin molecules in the precipitate and 40% in solution retain their ability for protein-protein interactions. This means that a polycation-bound enzyme is localized in the hydrophobic nucleus of the complex, whereas the polyanion-bound enzyme sits on the hydrophilic shell of the complex. On pH-induced phase transition (pH decreases from 6.1 to 5.7), there occurs a stepwise decrease in penicillin amidase activity which is due to a 9.8-fold increase in the Km for 2-nitro-4-phenylacetamidobenzoic acid. Change of the catalytic activity and thermal stability of N-PEC-bound penicillin amidase is fully reversible and reproducible. Such soluble-insoluble immobilized enzymes with controllable thermal stability and activity may be used for simulating events in vivo and in biotechnology.     

Involvement of active site in self-association of proteins: a case of alpha-chymotrypsin.

The self-association of proteolytic enzymes can be looked upon as an interesting possibility of the manifestation of enzyme-substrate complex. Hence the involvement of active site in such processes is a centre of investigation for many years. In the case of alpha-chymotrypsin, considerable controversy exists with regard to the involvement of active site of the enzyme in its self-association. A historical perspective of the problem and an overview of the available evidence, for and against, is presented and critically analysed. Despite contradicting observations, accumulated evidence indicates that His-57 and Ser-195 at the active site are involved, at least partially, in the self-association; a few other groups such as Tyr-146 and Met-192 are also involved in such processes.     

Hepatic microsomal epoxide hydrase. Involvement of a histidine at the active site suggests a nucleophilic mechanism

The effects of a wide variety of chemical modification reagents on the activity of purified rat liver microsomal epoxide hydrase have been investigated. Alkylating agents, such as the phenacyl bromides and benzyl bromide are potent inhibitors of epoxide hydrase. 2-Bromo-4'-nitroacetophenone (p-nitrophenacyl bromide) specifically and irreversibly inactivates epoxide hydrase. Pseudo-first order kinetics of inhibition is observed at higher inhibitor/enzyme ratios. The rate of inactivation is controlled by a group on the enzyme with an apparent pKa of 7.6. Inactivation of the enzyme with 14C-labeled 2-bromo-4'-nitroacetophenone leads to the incorporation of approximately 1 mol of radioactive inhibitor/mol of protein. Epoxide hydrase can be protected against this inactivation by the substrate phenanthrene-9,10-oxide. These results are consistent with the interpretation that 2-bromo-4'-nitroacetophenone acts as an active site-directed inhibitor. The site of alkylation by 2-bromo-4'-nitroacetophenone is a histidine residue of epoxide hydrase. The N-alkylated histidine derivative has been identified as 1-(p-nitrophenacyl)-4-histidine. A possible mechanism for the enzymatic hydration catalyzed by epoxide hydrase is discussed which involves a histidine residue of the enzyme serving as a general base catalyst for the nucleophilic addition of water.     

2,3-butanedione as a photosensitizing agent: application to alpha-amino acids and alpha-chymotrypsin

2,3-Butanedione sensitized the rapid photodestruction of free alpha-amino acids, and the photoinactivation of alpha-chymotrypsin, in the presence of ultraviolet light and oxygen. These reactions showed "pseudo-first-order" kinetics at 2,3-butanedione concentrations approximating those employed for the chemical modification of arginine residues in proteins. The photoreactions were inhibited in anoxic media or in the presence of azide; findings were consistent with a singlet oxygen mechanism for these reactions. No enhancement in the rate of reaction was observed in D2O. The rate of 2,3-butanedione-sensitized photodestruction of free amino acids increased with increasing pH. However, the rate constants for the photosensitized inactivation of alpha-chymotrypsin, as well as those for the photodestruction of the tryptophan residues of this enzyme, decreased linearly with increasing pH.     

The role of tryptophan residues in heparin-antithrombin interactions

A single tryptophan residue on antithrombin has been modified with dimethyl-(2-hydroxy-5-nitrobenzyl)sulfonium bromide. This alteration led to a 500-fold reduction in the heparin-dependent acceleration of thrombin-modified antithrombin interactions, as well as a 10-fold decrease in the avidity of the modified protease inhibitor for mucopolysaccharide. Preincubation of antithrombin with the octasaccharide binding domain of heparin prior to treatment with dimethyl-(2-hydroxy-5-nitrobenzyl)sulfonium bromide was able to suppress modification of the critical tryptophan and preserve the functional capacities of the protease inhibitor. Fluorescence quenching experiments indicated that the modifiable tryptophan groups of antithrombin were exposed to the solvent environment. Based upon these data, it was proposed that the loss of “heparin cofactor” activity of antithrombin must be predominantly due to an inability of the modified protease inhibitor to undergo a conformational transition required for mucopolysaccharide-dependent “activation” of the macromolecule.     

Physico-chemical properties of terrylytin modified by a copolymer based on vinylpyrrolidone

The proteolytic activity of terrylytin produced by the culture of Asp. terricola and modified by a water-soluble copolymer of vinylpyrrolidone and acrolein remained unchanged after enzyme modification. Using micro-thin layer chromatography, it was shown that the bulk of the epsilon-amino groups of lysine residues of the protein enter the reaction with the aldehyde groups of the polymeric matrix. The sedimentation and diffusion patterns of the polymerenzyme adduct demonstrated that the molecular weight of the modified enzyme is the total of molecular weights of its constituent components. Evidence from viscosimetry and gel chromatography allowed to develop a hydrodynamic model of the macromolecular product. It was shown that the rate of the enzyme inactivation in the solution calculated from the first order reaction equation depends on the nature of the enzyme electrochemical microenvironment. Under conditions close to physiological ones the rate inactivation constant for terrylytin modified by a neutral polymeric matrix is 10 times less than that for the native enzyme. At the isoelectric point (pH 4,6) a positively charged polymeric form of terrylytin is found to be the most stable one. The pH and temperature optima for casein hydrolysis remained unchanged throughout polymeric modification. The polymeric membrane did not hamper the diffusion during approximation of the substrates (casein and insulin) to the enzyme molecule during the catalytic act, which manifested itself in a constancy of Michaelis curves. Terrylytin modification by a copolymer causes an increase of stability with respect to trypsin proteolysis and a decrease of human blood plasma affinity for the inhibitors. The apparent inhibition constants for modified enzyme forms do not depend on the nature of electrochemical microenvironment and exceed that for native terrylytin 10-fold.