Structure and activity of trypsin in reverse micelles

The kinetic properties of trypsin have been studied in reverse micelles formed by two surfactant systems, namely bis(2-ethylhexyl) sodium sulfosuccinate (AOT) in isooctane, and hexadecyltrimethyl ammonium bromide (CTAB) in chloroform/isooctane (1:1, by vol.). Three substrates have been used, namely N alpha-benzoyl-L-Arg ethyl ester, N alpha-benzoyl-L-Phe-L-Val-L-Arg p-nitroanilide (BzPheValArg-NH-Np) in AOT and N alpha-benzyloxycarbonyl-L-Lys p-nitrophenyl ester (ZLysO-Np) in CTAB. One of the main aims of the work was to compare the behaviour of trypsin in reverse micelles with that of alpha-chymotrypsin, for which an enhancement of kcat had been observed with respect to aqueous solutions. The pH profile is not significantly altered in reverse micelles with respect to water, however the kinetic parameters (kcat and Km) differ widely from one another, and are markedly affected by the micellar conditions, in particular by the water content wo (wo = [H2O]/[AOT]). Whereas in the case of BzPheValArg-NH-Np kcat is much smaller than in water, in the case of ZLysO-Np at pH 3.2 (but not at pH 6.0) a slight enhancement with respect to water is observed. On the basis of rapid kinetic spectrophotometry (stopped-flow) and solvent isotope effect studies, this enhancement is ascribed to a change in the rate-limiting step (acylation rather than hydrolysis). As in the case of alpha-chymotrypsin, the maximal activity is found for all substrates at rather small wo values (below 12), which is taken to suggest that the enzyme works better when is surrounded by only a few layers of tightly bound water. Spectroscopic studies [ultraviolet absorption, circular dichroism (CD) and fluorescence] have been carried out as a function of wo. Whereas the absorption properties are practically unchanged, the CD spectrum in AOT micelles has a lower intensity than in water, which is interpreted as a partial unfolding. The intensity is partly restored when Ca2+ ions are added, indicating that the micellar environment may cause a partial denaturation by depleting it of calcium ions. Fluorescence data show that the emission properties of the protein in reverse micelles match those in aqueous solution at around wo = 13 approx., whereas lambda max shifts towards the red by increasing wo, indicating an exposure of the tryptophan residues and probably an unfolding of the whole protein, at wo values above 15. Finally the reaction between trypsin and its specific macromolecular Kunitz inhibitor from soybeans is studied.(ABSTRACT TRUNCATED AT 400 WORDS)     

Benzyloxycarbonylphenylalanylcitrulline p-nitroanilide as a substrate for papain and other plant cysteine proteinases.

After preliminary assays, with papain, bromelain and ficin, on a range of citrulline p-nitroanilides, values of Km and kcat. for the papain-catalysed hydrolysis of three derivatives, N alpha- benzyloxycarbonylcitrulline p-nitroanilide, benzyloxycarbonylphenylalanylcitrulline p-nitroanilide and benzyloxycarbonylglycylphenylalanylcitrulline p-nitroanilide, were obtained. It is concluded that benzyloxycarbonylphenylalanylcitrulline p-nitroanilide is a highly selective substrate for the sensitive detection and assay of the plant cysteine proteinases.     

Specific inhibition of human granulocyte elastase with peptide aldehydes

The kinetic features of human granulocyte elastase, chymotrypsin, porcine pancreatic elastase and elastomucoproteinase were compared. Amino acyl ester substrates were assayed and Km and kcat values were defined. Aldehyde analogues of the p-nitroanilide substrates designed for granulocyte elastase as optimal for Km appeared to be potent inhibitors. Suc-D-Phe-Pro-valinal (Ki = 40 microM) was found to inhibit granulocyte elastase competitively and specifically when measured with synthetic substrates, and the Ki was 3 microM with the natural protein substrate, elastin.     

Kinetics of hydrolysis of amide and anilide substrates of p-guanidino-L-phenylalanine by bovine and porcine trypsins

The rates of hydrolysis of N alpha-benzoyl-p-guanidino-L-phenylalaninamide (Bz-GPA-NH2) and N alpha-substituted p-nitroanilides (pNA) of GPA (benzyloxycarbonyl(Z)-GPA-pNA, benzoyl(Bz)-GPA-pNA and acetyl(Ac)-GPA-pNA) by bovine and porcine trypsins were compared with those of arginine (Arg) substrates. The amide type substrates of GPA were hydrolyzed as fast as those of Arg by the two enzymes with much the same kcat/Km values, though significant differences were found between the kcat and Km values of GPA derivatives and those of Arg derivatives. The kinetic behavior of porcine trypsin toward GPA substrates was almost the same as that of the bovine enzyme. The ratio of the kcat value for Bz-GPA-OEt to that for Bz-GPA-NH2 was much larger than that for the ester to amide substrates of arginine, suggesting that the conformational change of the active site of trypsin induced by a benzene ring in the side chain of Bz-GPA-OEt specifically increases the velocity of the deacylation process of the ester substrate. Remarkably low values of both kcat and Km were found for the tryptic hydrolysis of Z-GPA-pNA and Ac-GPA-pNA, as well as on that of Bz-GPA-pNA (Tsunematsu, H., et al. (1983) J. Biochem. 94, 123-128). Z-GPA-pNA is the best substrate for the two trypsins among the three N alpha-substituted anilide substrates of GPA.(ABSTRACT TRUNCATED AT 250 WORDS)     

A procedure for the joint evaluation of substrate partitioning and kinetic parameters for reactions catalyzed by enzymes in reverse micellar solutions. I. Hydrolysis of 2-naphthyl acetate catalyzed by lipase in sodium 1,4-bis(2-ethylhexyl) sulphosuccinate (AOT)/buffer/heptane

A simple method useful for the joint evaluation of substrate partitioning and kinetic parameters for reactions catalyzed by enzymes entrapped in reverse micelles is proposed. The method is applied to the hydrolysis of 2-naphthyl acetate (2-NA) catalyzed by lipase in sodium 1,4-bis(2-ethylhexyl) sulfosuccinate (AOT)/buffer/heptane reverse micellar solutions. In the presence of micelles, the relationship between the initial reaction rate and the analytical concentration of 2-NA was dependent on AOT concentration at a constant W ([water]/[AOT]) value. The dependence of the initial reaction rate profiles with [AOT] was analyzed according with the method proposed to obtain the partition constant of 2-NA between the micelles and the external solvent, Kp. A value of Kp = 2.7 L mol(-1) was obtained irrespective of the water content of the micelles (W from 5 to 20). The catalytic rate constant kcat in the micellar solutions was independent of [AOT] but slightly decreased with an increase in W from 2 x 10(-6) mol g(-1) s(-1) at W = 5 to 1.2 x 10(-6) mol g(-1) s(-1) at W = 20. The apparent Michaelis constant determined in terms of the analytical concentration of 2-NA increased with [AOT] at a given W and moderately decreased with W at a fixed [AOT]. The increase with [AOT] is accounted for by considering the partitioning of the substrate. After correction for the partitioning of 2-NA values of (Km)corr were obtained as 3.9 x 10(-3) mol L(-1) (W = 5), 4.6 x 10(-3) mol L(-1) (W = 10), 2.3 x 10(-3) mol L(-1) (W = 15), and 1.7 x 10(-3) mol L(-1) (W = 20). The rate parameters in the aqueous phase in the absence of micelles, were obtained as (kcat)aq = 7.9 x 10(-6) mol g(-1) s(-1) and (Km)aq = 2.5 x 10(-3) mol L(-1). In order to compare the efficiency of the enzyme in the micellar solution with that in aqueous phase, the values of (Km)corr were in turn corrected to take into account differences in the substrate activity, obtaining so a set of (Km)*corr values. The efficiency of the enzyme in the micellar solution, defined as the ratio, kcat/(Km)*corr, was found to be higher than in the aqueous phase, even at high water contents (W = 20). This higher efficiency is due to a significant decrease in (Km)*corr values.     

Kinetic behaviour of alpha-chymotrypsin in reverse micelles. A stopped-flow study.

At the aim of investigating whether the early rapid phase of enzyme turnover is different in reverse micelles compared with bulk water, the kinetic properties of alpha-chymotrypsin have been studied in reverse micelles formed by sodium bis(2-ethylhexyl)sulfosuccinate in isooctane. Pre-steady state and steady-state kinetic constants, in water and in reverse micelles, have been determined by stopped-flow spectrophotometry for the hydrolysis of two substrates, namely acetyl-L-tryptophan-p-nitrophenyl ester and p-nitrophenyl acetate. It has been shown that, for both substrates, the acylation rate constant (k2) is very much lower in reverse micelles than in water. However, the deacylation rate constant (k3) and the turnover number (kcat) are not significantly changed in reverse micelles with respect to bulk water. Therefore, despite considerable rate changes in the acylation step, deacylation is rate limiting both in water as well as in reverse micelles, under the experimental conditions used.     

Chemically stabilized trypsin used in dipeptide synthesis

Bovine pancreatic trypsin was treated with ethylene glycol bis(succinic acid N-hydroxysuccinimide ester). Approximately 8 of 14 lysines per trypsin molecule were modified. This derivative (EG trypsin) was more stable than native between 30 degrees and 70 degrees C: T50 values were 59 degrees C and 46 degrees C, respective. EG trypsin's half-life of 25 min at 55 degrees C was fivefold greater than native's. EG trypsin had a decreased rate of autolysis and retained more activity in aqueous mixtures of 1,4-dioxan, dimethylformamide, dimethylsulfoxide, and acetonitrile. EG trypsin had lower Km values for both amide and ester substrates; its kcat values for two amides (benzoyl-L-arginine p-nitroanilide and benzyloxycarbonyl glycyl-glycyl-arginyl-7-amino-4-methyl coumarin) increased, whereas its kcat value for an ester (thiobenzoyl benzoyloxycarbonyl-L-lysinate) decreased slightly. The specific activity (kcat/Km) of EG trypsin was increased for both amide and ester substrates. EG trypsin gave higher yields and reaction rates than native in kinetically controlled synthesis of benzoyl argininyl-leucinamide in acetonitrile and in t-butanol. Highest peptide yields occurred with EG trypsin in 95% acetonitrile, where 90% of the substrate was converted to product. No peptide synthesis occurred in 95% DMF with either form of trypsin.     

Immobilized enzymes in reverse micelles: studies with gel-entrapped trypsin and alpha-chymotrypsin in AOT reverse micelles

Trypsin and alpha-chymotrypsin were immobilized by gelentrapment in polyacrylamide cross-linked with N,N(1)-methylenebisacrylamide. The immobilized enzymes are catalytically efficient in suspensions of reverse micelles formed in isooctane by bis(2-ethylhexyl) sodium sulfosuccinate (AOT) and water. Both entrapped enzymes are stable in reverse micellar suspension at room temperature and pH 8.2 for 3 days and lose 30-40% activity after 1 week. The enzymes obey Michaelis-Menten kinetics in the investigated concentration range with K(m) values higher than those in solution. Activity of the enzymes is independent of the water content of the micellar solution. No shift in pH optimum was observed for immobilized trypsin activity toward Nalpha-benzoyl-L-arginine ethyl ester. The utility of the procedure, which combines the advantage of enzyme immobilization and enzymology in reverse micelles, is illustrated by an example of peptide synthesis. In particular, peptide synthesis (e. g., Z--Ala--Phe--Leu--NH(2)) using water-insoluble substrate has been performed with gelentrapped alpha-chymotrypsin in reverse micellar suspension with the advantage of efficient enzyme recycling.     

Activity and stability of horse-liver alcohol dehydrogenase in sodium dioctylsulfosuccinate/cyclohexane reverse micelles

Horse liver alcohol dehydrogenase (EC 1.1.1.1) solubilized in sodium dioctylsulfosuccinate (AOT)/cyclohexane reverse micelles was used for the oxidation of ethanol and reduction of cyclohexanone in a coupled substrate/coenzyme recycling system. The activity of the enzyme was studied as a function of pH and water content. The enzyme was optimally active in microemulsions prepared with buffer of pH around 8. An increase in enzymatic activity was observed as a function of increasing water content. The Km values for the substrates were calculated based on the total reaction volume. The apparent Km for ethanol in reverse micelles was about eight times lower as compared to that in buffer solution, whereas the Km for cyclohexanone was almost unaltered. Storage and operational stability were investigated. It was found that the specific activity of the alcohol dehydrogenase operating in reverse micellar solution was good for at least two weeks. The steroid eticholan-3 beta-ol-17-one was also used as a substrate. In this case the reaction rate was approximately five times higher in a reverse micellar solution than in buffer.     

Dependence of the catalytic activity of papain on the ionization of two acidic groups.

The pH dependence of kcat/Km for the papain-catalyzed hydrolysis of ethyl hippurate, N-alpha-benzoyl-L-citrulline methyl ester, and the p-nitroanilide, amide, and ethyl ester derivatives of N-alpha-benzoyl-L-arginine was determined below pH 6.4. The value of kcat/Km was observed to be modulated by two acid ionizations rather than a single ionization as previously believed. For the five substrates studied, the average pK values for the two ionizations are 3.78 +/- 0.2 and 3.95 +/- 0.1 at T/2 0.3, 25 degrees C. The observation that similar pK values were obtained with different substrates was taken as evidence that the kinetically determined pK values are close in value to true macroscopic ionization constants for ionization of groups on the free enzyme.     

Sensitive assay of cysteine proteinases using new peptide p-nitroanilides

N-Succinyl-glycyl-leucyl-cystein(S-benzyl) p-nitroanilide and N-succinyl-leucyl-leucyl-cystein(S-benzyl) p-nitroanilide were found to be very sensitive substrates for the assay of papain, ficin, and bromelain. These p-nitroanilides were hydrolyzed only very slightly by chymotrypsin, but not detectably by trypsin.     

Mechanistic studies on thrombin catalysis

The kinetic mechanism of the cleavage of four p-nitroanilide (pNA) substrates by human alpha-thrombin has been investigated by using a number of steady-state kinetic techniques. Solvent isotope and viscosity effects were used to determine the stickiness of the substrates at the pH optimum of the reaction; a sticky substrate is defined as one that undergoes catalysis faster than it dissociates from the Michaelis complex. Whereas benzoyl-Arg-pNA could be classified as a nonsticky substrate, D-Phe-pipecolyl-Arg-pNA was very sticky. The other two substrates (tosyl-Gly-Pro-Arg-pNA and acetyl-D-Phe-pipecolyl-Arg-pNA) were slightly sticky. The pH profiles of kcat/Km were bell-shaped for all substrates. The pKa values determined from the pH dependence of kcat/Km for benzoyl-Arg-pNA were about 7.5 and 9.1. Similar pKa values were determined from the pH profiles of kcat/Km for tosyl-Gly-Pro-Arg-pNA and acetyl-D-Phe-pipecolyl-Arg-pNA and for the binding of the competitive inhibitor N alpha-dansyl-L-arginine-4-methylpiperidine amide. The groups responsible for the observed pKa values were proposed to be His57 and the alpha-amino group of Ile16. The temperature dependence of the pKa values was consistent with this assignment. The pKa values of 6.7 and 8.6 observed in the pH profile of kcat/Km for D-Phe-pipecolyl-Arg-pNA were displaced to lower values than those observed for the other substrates. The displacement of the acidic pKa value could be attributed to the stickiness of this substrate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modified activity of Aeromonas aminopeptidase: metal ion substitutions and role of substrates

Aeromonas aminopeptidase contains two nonidentical metal binding sites that have been shown by both spectroscopy and kinetics to be capable of interacting with one another [Prescott, J.M., Wagner, F.W., Holmquist, B., & Vallee, B.L. (1985) Biochemistry 24, 5350-5356]. The effects of metal ion substitutions on the susceptibility of the p-nitroanilides of L-alanine, L-valine, and L-leucine--substrates that are hydrolyzed at widely differing rates by native Aeromonas aminopeptidase--were studied by determining values of kcat and Km for the 16 metalloenzymes that result from all possible combinations of Zn2+, Co2+, Ni2+, and Cu2+ in each of the two sites. The different combinations of metal ions and substrates yield a broad range in kinetic values; kcat varies by more than 1800-fold, Km by 3000-fold, and kcat/Km ratios by more than 10,000. L-Leucine-p-nitroanilide is by far the most susceptible of the three substrates, and the hyperactivation previously observed with aminopeptidase containing either Ni2+ or Cu2+ in the first binding site and Zn2+ in the second site occurs only with the two poorer substrates, L-alanine-p-nitroanilide and L-valine-p-nitroanilide. Although the enzyme with Zn2+ in both sites hydrolyzes the substrates with N-terminal alanine and valine poorly, it is extremely effective toward L-leucine-p-nitroanilide. Neither metal binding site can be identified as controlling either Km or kcat; both parameters are influenced by the identity of the metal ions, by the site each occupies, and, most strongly, by the substrate.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of side chain structure of ester substrates in determining the rate-controlling step in alpha-chymotrypsin-catalyzed hydrolysis

Presteady state and steady state analyses of the alpha-chymotrypsin [EC 3.4.21.1]-catalyzed hydrolysis of three specific ester substrates and three ring-substituted derivatives were carried out to elucidate the effect of hydrophobic interactions due to the different side chains of the substrates on the individual steps of the reaction. Hydrolysis of all the substrates except for N alpha-acetyl-Nin-formyltryptophan methyl ester (Ac-Trp(CHO)-OMe) was controlled by the deacylation rate. In spite of their comparable Ks values, the substrates with small kcat, such as N alpha-acetyltryptophan methyl ester and N alpha-acetyl-2-(2-nitro-4-carboxyphenylsufenyl)-tryptophan methyl ester, characteristically gave Km values one order of magnitude smaller than the others. For the reaction of Ac-Trp(CHO)-OMe, it was ascertained that the deacylation step was not rate-controlling. It is suggested that the acylation step controls the rate in this case.     

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.     

The specificity of two proteinases that cleave adjacent to arginine, C1 esterase and acrosin, for peptide p-nitroanilide substrates

Relative values of Vmax/Km for hydrolysis of 40 peptide p-nitroanilides catalyzed by human Cl-s and human acrosin are reported. For Cl-s, Ac-Lys(gamma Cbz)-Gly-Arg is the optimum sequence, but 25% of the substrates have (Vmax/Km)rel greater than 0.25 compared to this sequence. The best acrosin substrate tested has the sequence Tos-Gly-Pro-Arg, although (Vmax/Km)rel greater than 0.15 for more than half of the substrates. Proline at P2 is preferred by acrosin. Both enzymes prefer arginine at P1 greater than or equal to 3-fold over lysine and will not accept citrulline. In addition, occupancy of site S3 may yield an increase in Vmax/Km of greater than or equal to 10-fold with either enzyme, but many residues are accepted at S2, S3 and S4. Thus, an acrosin assay using Tos-Gly-Pro-Arg p-nitroanilide as a substrate is more than 20-times as sensitive as existing assays with blocked arginine derivatives.     

Substituent effects on substrate activation and Michaelis-Menten Kinetic parameters in the alpha-chymotrypsin-catalyzed hydrolysis of phenyl acetates.

The effects of substituents on the steady state and pre-steady state kinetics in alpha-chymotrypsin [EC 3.4.21.1]-catalyzed hydrolysis were studied using substituted phenyl acetates. In the steady state hydrolysis, substrate activation, which had been observed and studied previously for p-nitrophenyl acetate, was also observed for p-bromo, p-chloro-, and m-methylphenyl acetates. Little activation was observed for p-acetyl-, m-nitro-, p-methyl-, and p-methoxyphenyl acetates. Addition of p-dichlorobenzene increased kcat for all substrates examined and greatly diminished the substrate activation for the activatable substrate(s) to activator binding site(s). The value of kcat decreased in accordance with increase of the sigma-value of substituents. On the other hand, kcat/Km (app) showed an opposite sigma- dependence, as was previously observed. In pre-steady state measurements, little burst was observed for more electron-donating substituents than m-nitro. The sigma dependence of kcat is apparently not consistent with the prediction derived from that of kcat/Km (app) on the basis of the usual two-step mechanism with a common acetyl-enzyme intermediate.     

Rapid optimization of enzyme substrates using defined substrate mixtures.

A strategy is described for the rapid optimization of kcat/Km for protease substrates. Selected positions of a given peptide substrate sequence are varied through synthesis with mixtures of amino acids. Incubation of the resulting peptide mixture with the enzyme of interest and analysis by high pressure liquid chromatography provides a direct measure of analogs with enhanced kcat/Km. High performance liquid chromatography/continuous flow fast atom bombardment mass spectrometry is used to assign structure to each peak in the chromatogram. As an example of the utility and efficiency of "substrate mapping" we describe optimization of the collagenase substrate Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 (where Dnp is dinitrophenyl) at the P'1 and P'2 positions. Six different mixtures were prepared for evaluation, representing the synthesis of 128 different synthetic substrates. "Substrate mapping" has led to Dnp-Pro-Leu-Gly-Cys(Me)-His-Ala-D-Arg-NH2, a substrate that possesses a 10-fold better kcat/Km than Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2.     

Conformation aspects of peptide interaction with proteolytic enzymes. Alpha-chymotrypsin catalyzed hydrolysis of the cyclopeptides containing leucyltyrosyl fragments]

The studies were made on the interaction of alpha-chymotrypsin with a series of cyclopeptides cyclo(-L-leucyl-L-tyrosyl-glycyln-), n=4, 6 and 8 (I, II and III respectively), and cyclo(-L-leucyl-L-tryosyl-beta-aminovalero-yl2-) (IV). Compounds I and IV are resistant to enzyme action whereas cyclopeptides II and III proved to be the substrates, their kinetic constants being Km=15.4 and 13.2 mM and kcat=0.54 and 9.53 sec-1 respectively. The binding capacity of cyclopeptides I-IV is evaluated by their competitive inhibition of alpha-chymotrypsin catalyzed hydrolysis of N-acetyl-L-tyrosine methyl ester.     

Interactions of derivatives of guanidinophenylalanine and guanidinophenylglycine with Streptomyces griseus trypsin

The rates of hydrolysis of the ester, amide and anilide substrates of p-guanidino-L-phenylalanine (GPA) by Streptomyces griseus trypsin (S. griseus trypsin) were compared with those of arginine (Arg) substrates. The specificity constant (kcat/km) for the hydrolysis of GPA substrates by the enzyme was 2-3-times lower than that for arginine substrates. The kcat and Km values for the hydrolysis of N alpha-benzoyl-p-guanidino-L-phenylalanine ethyl ester (Bz-GPA-OEt) by S. griseus trypsin are in the same order of magnitude as those of N alpha-benzoyl-L-arginine ethyl ester (Bz-Arg-OEt), although both values for the former when hydrolyzed by bovine trypsin are higher by one order of magnitude than those for the latter. The specificity constant for the hydrolysis of Bz-GPA-OEt by S. griseus trypsin is much higher than that for N alpha-benzoyl-p-guanidino-L-phenylglycine ethyl ester (Bz-GPG-OEt). As with the kinetic behavior of bovine trypsin, low values in Km and kcat were observed for the hydrolysis of amide and anilide substrates of GPA by S. griseus trypsin compared with those of arginine substrates. The rates of hydrolysis of GPA and arginine substrates by S. griseus trypsin are about 2- to 62-times higher than those obtained by bovine trypsin. Substrate activation was observed with S. griseus trypsin in the hydrolysis of Bz-GPA-OEt as well as Bz-Arg-OEt, whereas substrate inhibition was observed in three kinds of N alpha-protected anilide substrates of GPA and arginine. In contrast, no activation by the amide substrate of GPA could be detected with this enzyme.