Use of a fluorescence plate reader for measuring kinetic parameters with inner filter effect correction

A general method is presented here for the determination of the Km, kcat, and kcat/Km of fluorescence resonance energy transfer (FRET) substrates using a fluorescence plate reader. A simple empirical method for correcting for the inner filter effect is shown to enable accurate and undistorted measurements of these very important kinetic parameters. Inner filter effect corrected rates of hydrolysis of a FRET peptide substrate by hepatitis C virus (HCV) NS3 protease at various substrate concentrations enabled measurement of a Km value of 4.4 +/- 0.3 microM and kcat/Km value of 96,500 +/- 5800 M-1 s-1. These values are very close to the HPLC-determined Km value of 4.6 +/- 0.7 microM and kcat/Km value of 92,600 +/- 14,000 M-1 s-1. We demonstrate that the inner filter effect correction of microtiter plate reader velocities enables rapid measurement of Ki and Ki' values and kinetic inhibition mechanisms for HCV NS3 protease inhibitors.     

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.     

Replacement of an interdomain residue Val39 of Escherichia coli aspartate aminotransferase affects the catalytic competence without altering the substrate specificity of the enzyme

Three mutant Escherichia coli aspartate aminotransferases in which Val39 was changed to Ala, Leu, and Phe by site-directed mutagenesis were prepared and characterized. Among the three mutant and the wild-type enzymes, the Leu39 enzyme had the lowest Km values for dicarboxylic substrates. The Km values of the Ala39 enzyme for dicarboxylates were essentially the same as those of the wild-type (Val39) enzyme. These two mutant enzymes showed essentially the same kcat values for dicarboxylic substrates as did the wild-type enzyme. On the other hand, incorporation of a bulky side-chain at position 39 (Phe39 enzyme) decreased both the affinity (1/Km) and catalytic ability (kcat) toward dicarboxylic substrates. These results show that the position 39 residue is involved in the modulation of both the binding of dicarboxylic substrates to enzyme and the catalytic ability of the enzyme. Although the replacement of Val39 with other residues altered both the kcat and Km values toward various substrates including dicarboxylic and aromatic amino acids and the corresponding oxo acids, it did not alter the ratio of the kcat/Km value of the enzyme toward a dicarboxylic substrate to that for an aromatic substrate. The affinity for aromatic substrates was not affected by changing the residue at position 39. These data indicate that, although the side chain bulkiness of the residue at position 39 correlates well with the activity toward aromatic substrates in the sequence alignment of several aminotransferases [Seville, M., Vincent M.G., & Hahn, K. (1988) Biochemistry 27, 8344-8349], the residue does not seem to be involved in the recognition of aromatic substrates.     

Subsite mapping of an acidic amino acid-specific endopeptidase from Streptomyces griseus, GluSGP, and protease V8.

The substrate specificities of an acidic amino acid-specific endopeptidase of Streptomyces griseus, GluSGP, and protease V8 [EC 3.4.21.19] were investigated with peptide p-nitroanilide substrates which have a Glu residue at the P1 position. GluSGP and protease V8 favored Pro and Leu residues at S2, respectively, while the S3 subsite of GluSGP preferred Phe over either Ala or Leu. The S3 subsite of protease V8 preferred Leu over either Ala or Phe. The best substrates for GluSGP and for protease V8 were Boc-Ala-Phe-Pro-Glu-pNA with a Km value of 0.41 mM (0.1 M Tris-HCl, pH 8.8) and Boc-Ala-Leu-Leu-Glu-pNA with a Km value of 0.25 mM (0.1 M phosphate, pH 7.8), respectively. The kcat/Km values for these substrates obtained with GluSGP were about one hundred to twenty thousand times larger than those obtained with protease V8. Protease V8 exhibited a single optimal pH of around 8 for the hydrolysis of Boc-Ala-Ala-Leu-Glu-pNA and Boc-Ala-Leu-Leu-Asp-pNA.     

Human cathepsin H: deletion of the mini-chain switches substrate specificity from aminopeptidase to endopeptidase

The mini-chain of human cathepsin H has been identified as the major structural element determining the protease's substrate specificity. A genetically engineered mutant of human cathepsin H lacking the mini-chain, des[Glu(-18)-Thr(-11)]-cathepsin H, exhibits endopeptidase activity towards the synthetic substrate Z-Phe-Arg-NH-Mec (kcat = 0.4 s(-1), Km = 92 microM, kcat/Km = 4348 M(-1) s(-1)) which is not cleaved by r-wt cathepsin H. However, the mutant enzyme shows only minimal aminopeptidase activity for H-Arg-NH-Mec (kcat = 0.8 s(-1), Km = 3.6 mM, kcat/Km = 222 M(-1) s(-1)) which is one of the best known substrates for native human cathepsin H (kcat = 2.5 s(-1), Km = 150 microM, kcat/Km = 16666 M(-1) s(-1)). Inhibition studies with chicken egg white cystatin and E-64 suggest that the mini-chain normally restricts access of inhibitors to the active site. The kinetic data on substrates hydrolysis and enzyme inhibition point out the role of the mini-chain as a structural framework for transition state stabilization of free alpha-amino groups of substrates and as a structural barrier for endopeptidase-like substrate cleavage.     

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 investigation of the staphylococcal protease-catalyzed hydrolysis of synthetic substrates.

In investigating the staphylococcal protease-catalyzed hydrolysis of N-tert-butoxycarbonyl-L-glutamate alpha-phenyl ester, N-benzyloxycarbonyl-L-glutamate alpha-phenyl ester and N-benzyloxycarbonyl-L-glutamate alpha-p-nitroanilide, we obtained kinetic evidence consistent with the formation of an acyl-enzyme intermediate. We found that addition of a nucleophile, such as methanol, led to the partition of the common acyl-enzyme intermediate between water and the alcohol. With N-benzyl-oxycarbonyl-L-glutamate alpha-phenyl ester, a specific ester substrate, deacylation was shown to be the rate-limiting step. By studying the kcat/Km ratio of these hydrolyses as a function of pH, we have shown that two ionizable groups on the enzyme are essential to the catalytic process. One of these groups has a pK of 6.58 and the other, a pK of 8.25. The assignment of these pK values is discussed in connection with the known features of the serine proteinase reaction mechanism. In addition, monovalent anions were shown to inhibit staphylococcal protease hydrolyses. They seem to compete with the negative charge of the substrate, thus inhibiting its binding on the enzyme molecule. Finally we compared the kinetic parameters obtained with five proteases isolated from different strains of Staphylococcus aureus.     

The ywad gene from Bacillus subtilis encodes a double-zinc aminopeptidase

The yet uncharacterized ywad gene from Bacillus subtilis has been cloned and overexpressed in Escherichia coli. The gene product (BSAP) was purified and shown to be an aminopeptidase. The activity of BSAP was optimal at pH 8.4, the enzyme was stable for 20 min at 80 degrees C and its activity was not affected by serine protease and aspartic protease inhibitors, but was completely diminished by the Zn-chelator 1,10-phenanthroline. ZnCl2 was able to restore activity, and the binding stoichiometry of zinc to apo-BSAP indicated two Zn ions per protein molecule. BSAP exhibited high preference toward p-nitroanilide derived Arg, Lys, and Leu synthetic substrates resulting in kcat/Km values of 1-5 x 10(1) s(-1) mM(-1).     

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)     

New, sensitive fluorogenic substrates for human cathepsin G based on the sequence of serpin-reactive site loops.

Cathepsin G has both trypsin- and chymotrypsin-like activity, but studies on its enzymatic properties have been limited by a lack of sensitive synthetic substrates. Cathepsin G activity is physiologically controlled by the fast acting serpin inhibitors alpha1-antichymotrypsin and alpha1-proteinase inhibitor, in which the reactive site loops are cleaved during interaction with their target enzymes. We therefore synthesized a series of intramolecularly quenched fluorogenic peptides based on the sequence of various serpin loops. Those peptides were assayed as substrates for cathepsin G and other chymotrypsin-like enzymes including chymotrypsin and chymase. Peptide substrates derived from the alpha1-antichymotrypsin loop were the most sensitive for cathepsin G with kcat/Km values of 5-20 mM-1 s-1. Substitutions were introduced at positions P1 and P2 in alpha1-antichymotrypsin-derived substrates to tentatively improve their sensitivity. Replacement of Leu-Leu in ortho-aminobenzoyl (Abz)-Thr-Leu-Leu-Ser-Ala-Leu-Gln-N-(2, 4-dinitrophenyl)ethylenediamine (EDDnp) by Pro-Phe in Abz-Thr-Pro-Phe-Ser-Ala-Leu-Gln-EDDnp produced the most sensitive substrate of cathepsin G ever reported. It was cleaved with a specificity constant kcat/Km of 150 mM-1 s-1. Analysis by molecular modeling of a peptide substrate bound into the cathepsin G active site revealed that, in addition to the protease S1 subsite, subsites S1' and S2' significantly contribute to the definition of the substrate specificity of cathepsin G.     

Interaction of thrombin des-ETW with antithrombin III, the Kunitz inhibitors, thrombomodulin and protein C. Structural link between the autolysis loop and the Tyr-Pro-Pro-Trp insertion of thrombin.

X-ray diffraction studies of human thrombin revealed that compared with trypsin, two insertions (B and C) potentially limit access to the active site groove. When amino acids Glu146, Thr147, and Trp148, adjacent to the C-insertion (autolysis loop), are deleted the resulting thrombin (des-ETW) has dramatically altered interaction with serine protease inhibitors. Whereas des-ETW resists antithrombin III inactivation with a rate constant (Kon) approximately 350-fold slower than for thrombin, des-ETW is remarkably sensitive to the Kunitz inhibitors, with inhibition constants (Ki) decreased from 2.6 microM to 34 nM for the soybean trypsin inhibitor and from 52 microM to 1.8 microM for the bovine pancreatic trypsin inhibitor. The affinity for hirudin (Ki = 5.6 pM) is weakened at least 30-fold compared with recombinant thrombin. The mutation affects the charge stabilizing system and the primary binding pocket of thrombin as depicted by a decrease in Kon for diisopropylfluorophosphate (9.5-fold) and for N alpha-p-tosyl-L-lysine-chloromethyl ketone (51-fold) and a 39-fold increase in the Ki for benzamidine. With peptidyl p-nitroanilide substrates, the des-ETW deletion results in changes in the Michaelis (Km) and/or catalytic (kcat) constants, worsened as much as 85-fold (Km) or 100-fold (kcat). The specific clotting activity of des-ETW is less than 5% that of thrombin and the kcat/Km for protein C activation in the absence of cofactor less than 2%. Thrombomodulin binds to des-ETW with a dissociation constant of approximately 2.5 nM and partially restores its ability to activate protein C since, in the presence of the cofactor, kcat/Km rises to 6.5% that of thrombin. This study suggests that the ETW motif of thrombin prevents (directly or indirectly) its interaction with the two Kunitz inhibitors and is not essential for the thrombomodulin-mediated enhancement of protein C activation.     

Substrate specificity of human cathepsin D using internally quenched fluorescent peptides derived from reactive site loop of kallistatin

Kallistatin, a serpin that specifically inhibits human tissue kallikrein, was demonstrated to be cleaved at the Phe-Phe bond in its reactive site loop (RSL) by cathepsin D. Internally quenched fluorescent peptides containing the amino acid sequence of kallistatin RSL were highly susceptible to hydrolysis by cathepsin D. Surprisingly, these peptides were efficiently hydrolyzed at Phe-Phe bond, despite having Lys and Ser at P2 and P2' positions, respectively, which was reported to be very unfavorable for substrates for cathepsin D. Due to the importance of cathepsin D in several physiological and pathological processes, we took the peptide containing kallistatin RSL sequence, Abz-Ala-Ile-Lys-Phe-Phe-Ser-Arg-Gln-EDDnp, as a reference substrate for a systematic specificity study of S3 to S3' protease subsites (EDDnp=N-[2,4-dinitrophenyl]-ethylenediamine and Abz=ortho-amino benzoic acid). We present in this paper some internally quenched fluorescent peptides that were efficient substrates for cathepsin D. They essentially differ from other previously described substrates by their higher kcat/Km values due, mainly, to low Km values, such as the substrate Abz-Ala-Ile-Ala-Phe-Phe-Ser-Arg-Gln-EDDnp (Km=0.27 microM, kcat=16.25 s(-1), kcat/Km=60185 microM(-1) x s(-1)).     

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.     

Plasminogen activation by single-chain urokinase in functional isolation. A kinetic study

The kinetics of the activation of Glu- and Lys-plasminogen by single-chain urokinase (sc urokinase) derived from the transformed human kidney cell line TCL-598 have been studied and compared with two-chain urokinase (tc urokinase). Plasminogen activation was determined by the increase in fluorescence polarization of fluorescein-labeled aprotinin, a high affinity inhibitor of plasmin. This methodology allows plasmin generation by sc urokinase to be measured in functional isolation, with no interfering generation of tc urokinase, sc urokinase was found to activate plasminogen to plasmin with apparent Michaelis-Menten-type kinetics. The Km for Glu-plasminogen activation was 47.7 microM, with a catalytic constant of 2.91 min-1. Lys-plasminogen activation by sc urokinase was characterized by a Km of 11.7 microM and a kcat of 5.60 min-1. The Km values for the activation of Glu- and Lys-plasminogen by tc urokinase were found to be similar to those for activation by sc urokinase (36.8 and 9.0 microM, respectively), but the catalytic constants were higher at 36.0 and 118 min-1, respectively. Therefore, on the basis of the catalytic efficiency kcat/Km, sc urokinase seems to have 16-27-fold lower activity than tc urokinase. This activity of sc urokinase is in contrast to its lack of activity against a low molecular weight peptide substrate (less than 0.2% of the activity of sc urokinase). The activation of sc urokinase to tc urokinase by plasmin was also characterized (Km = 3.0 microM, kcat = 105 min-1). Using these data, it was possible to calculate the theoretical rate of plasminogen activation by sc urokinase in the absence of aprotinin, when tc urokinase is generated by the action of plasmin. The calculated rate was in good agreement with that determined experimentally using the chromogenic substrate D-Val-Leu-Lys-p-nitroanilide. These data demonstrate that sc urokinase has properties which distinguish it from conventional serine protease zymogens. The lack of activity against low molecular weight peptide substrates demonstrates the inaccessibility of the substrate-binding pocket. However, there is a moderate activity against plasminogen, suggesting that plasminogen may be acting as both an effector and a substrate for sc urokinase.     

Properties of serine：glyoxylate aminotransferase purified from Arabidopsis thaliana leaves

The photorespiratory enzyme L-serine：glyoxylate amino- transferase （SGAT; EC 2.6.1.45） was purified from Arabidopsis thaliana leaves. The f＇mal enzyme was approximately 80 % pure as revealed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis with silver staining. The identity of the enzyme was confirmed by LC/MS/MS analysis. The molecular mass estimated by gel filtration chromato- graphy on Sephadex G-150 under non-denaturing conditions, mass spectrometry （matrix-assisted laser desorption/ ionization/time of flight technique） and sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 82.4 kDa, 42.0 kDa, and 39.8 kDa, respectively, indicating dimer as the active form. The optimum pH value was 9.2. The enzyme activity was inhibited by aminooxyacetate and β-chloro-L-alanine both compounds reacting with the carbonyl group of pyridoxal phosphate. The enzyme＇s transaminating activity with L-alanine and glyoxylate as substrates was approximately 55 % of that observed with L-serine and glyoxylate. The lower Kmvalue （1.25 mM） for L-alanine, compared with that of other plant SGATs, and the kcat/Km（Ala） ratio being approxi- mately 2-fold higher than kcat/Km（Ser） suggested that, during photorespiration, Ala and Ser are used by Arabidopsis SGAT with equal efficiency as amino group donors for glyoxylate. The equilibrium constant （Keq）, derived from the Haldane relation, for the transamination reaction between L-serine and glyoxylate with the formation of hydroxypyruvate and glycine was 79.1, strongly favoring glycine synthesis. However, it was accompanied by a low Km value of 2.83 mM for glycine. A comparison of some kinetic properties of the studied enzymes with the recombinant Arabidopsis SGATs previously obtained revealed substantial differences. The ratio of the velocity of the transamination reaction with L-alanine and glyoxylate as substrates versus that with L-serine and glyoxylate was 1：1.8 for the native enzyme, whereas it was 1：7 for the recombinant SGAT. Native SGAT showed a much lower Km value for L-alanine compared to the recombinant enzyme.     

Enkephalin hydrolysis by human serum biotinidase.

Purified human serum biotinidase exhibited amino-exo-peptidase activity. Enkephalins and dynorphin A (less than 10-mer) seemed to be the most appropriate substrates among various physiological peptides in terms of the kcat/Km values. Similar kcat/Km values were obtained for both biocytin (biotinyllysine) and these opioid-neuropeptides. Neuro-oligo-peptides ranging from 2-mer to 18-mer were hydrolyzed. The presence of amino group at the carboxyl terminal position increased the kcat/Km value by decreasing the Km value. The results of inhibition studies using various kinds of antibiotic inhibitors, metals, and chelating agents indicated that enkephalin hydrolysis was mediated by the peptide-hydrolyzing center probably containing Zn ions. This aminopeptidase activity was uniquely inhibited by a vitamin of biocytin. The reason for the high content of biotinidase activity in serum may be related to the binary function of this enzyme; i.e., biocytin hydrolyzing amidase and enkephalin hydrolyzing aminopeptidase functions.     

Mutant subtilisin E with enhanced protease activity obtained by site-directed mutagenesis

The specific activity of subtilisin E, an alkaline serine protease of Bacillus subtilis, was substantially increased by optimizing the amino acid residue at position 31 (Ile in the wild-type enzyme) in the vicinity of the catalytic triad of the enzyme. Eight uncharged amino acids (Cys, Ser, Thr, Gly, Ala, Val, Leu, and Phe) were introduced at this site, which is next to catalytic Asp32, using site-directed mutagenesis. Mutant enzymes were expressed in Escherichia coli and were prepared from the periplasmic space. Only the Val and Leu substitutions gave active enzyme, and the Leu31 mutant was found to have a greatly increased activity compared to the wild-type enzyme. The other six mutant enzymes showed a marked decrease in activity. This result indicates that a branched-chain amino acid at position 31 is essential for the expression of subtilisin activity and that the level of the activity depends on side chain structure. The purified Leu31 mutant enzyme was analyzed with respect to substrate specificity, heat stability, and optimal temperature. It was found that the Leu31 replacement caused a prominent 2-6-fold increase in catalytic efficiency (kcat/Km) due to a larger kcat for peptide substrates.     

Effectors of HIV-1 protease peptidolytic activity

High concentrations of salts dramatically affect the interaction of small ligands with HIV-1 protease. For instance, the Km and kcat values for Abz-Thr-Ile-Nle-p-nitro-Phe-Gln-Arg-NH2 (S) increased 120-fold and 3-fold, respectively, as the NaCl concentration in the assay decreased from 4.0 to 0.5 M. The Kd value for the competitive inhibitor amprenavir increased 12-fold over this concentration range of NaCl. The bimolecular rate constant for association of enzyme with amprenavir was independent of NaCl concentration, whereas the dissociation rate constant decreased with increasing NaCl concentration. Polyanionic polymers such as heparin or poly A substituted for NaCl. For example, the value of kcat/Km for S was 0.18 microM(-1) x s(-1) when the enzyme (<10 nM) was assayed in the standard buffer supplemented with 5 mM NaCl. If 0.01% poly A were included, the value of kcat/Km increased to 8.6 microM(-1) x s(-1). A DNA oligomer (23-mer) with an hexachlorofluoresceinyl moiety linked to the 5' end was studied as a model polyanionic polymer. The enzyme bound HF23 (Kd < 1 nM) with concomitant quenching of the hexachlorofluoresceinyl fluorescence. The stoichiometry for binding was 3 mol of enzyme per mol of oligomer. The hydrolytic activity of the enzyme with this oligomer was similar to that observed with poly A or high salt concentration when the molar ratio of oligomer to enzyme was greater than one. The results presented herein demonstrate that polyanionic polymers substitute for salts as effectors of HIV protease.     

Catalytic properties of porcine pancreatic elastase: a steady-state and pre-steady-state study

Pre-steady-state and steady-state kinetics for the p.p. elastase-catalysed hydrolysis of ZAlaONp, one of the most favourable substrates for this serine protease, have been studied between pH 4.0 and 8.0. The results are consistent with the minimum three-step mechanism: (formula; see text) Under pre-steady-state conditions, where [E0] much greater than [S0], the values of the dissociation constant of the E X S complex (Ks = k-1/k+1) and of the individual rate constants for the catalytic steps (k+2 and k+3) have been determined over the whole pH range explored. Under steady-state conditions, where [S0] much greater than [E0], the values of kcat and Km have been obtained over the same pH range. The pH profiles of k+2, k+3, k+2/Ks, kcat, kcat/Km reflect the ionization of a group, probably His57, with a pKa value of 6.85 +/- 0.10. The values of Ks and Km are pH independent. The steady-state parameters for the p.p. elastase-catalysed hydrolysis of a number of p-nitrophenyl esters of N-alpha-carbobenzoxy-L-amino acids have been also determined between pH 4.0 and 8.0 and compared with those of b.beta-trypsin and b.alpha-chymotrypsin. For all the substrates examined the acylation step (k+2) is rate limiting in the p.p. elastase catalysis, between pH 4.0 and 8.0. The different catalytic behaviours of p.p. elastase, b.beta-trypsin and b.alpha-chymotrypsin are consistent with the known three-dimensional structures of these serine proteases.     

Studies on the activity and activation of rat liver microsomal glutathione transferase with a series of glutathione analogues

The substrate specificity of rat liver microsomal glutathione transferase toward glutathione has been examined in a systematic manner. Out of a glycyl-modified and eight gamma-glutamyl-modified glutathione analogues, it was found that four (glutaryl-L-Cys-Gly, alpha-L-Glu-L-Cys-Gly, alpha-D-Glu-L-Cys-Gly, and gamma-L-Glu-L-Cys-beta-Ala) function as substrates. The kinetic parameters for three of these substrates (the alpha-D-Glu-L-Cys-Gly analogue gave very low activity) were compared with those of GSH with both unactivated and the N-ethylmaleimide-activated microsomal glutathione transferase. The alpha-L-Glu-L-Cys-Gly analogue is similar to GSH in that it has a higher kcat (6.9 versus 0.6 s-1) value with the activated enzyme compared with the unactivated enzyme but displays a high Km (6 versus 11 mM) with both forms. Glutaryl-L-Cys-Gly, in contrast, exhibited a similar kcat (8.9 versus 6.7 s-1) with the N-ethylmaleimide-treated enzyme but retains a higher Km value (50 versus 15 mM). Thus, the alpha-amino group of the glutamyl residue in GSH is important for the activity of the activated microsomal glutathione transferase. These observations were quantitated by analyzing the changes in the Gibbs free energy of binding calculated from the changes in kcat/Km values, comparing the analogues to GSH and each other. It is estimated that the binding energy of the alpha-amino group of the glutamyl residue in GSH contributes 9.7 kJ/mol to catalysis by the activated enzyme, whereas the corresponding value for the unactivated enzyme is 3.2 kJ/mol. The importance of the acidic functions in glutathione is also evident as shown by the lack of activity with 4-aminobutyric acid-L-Cys-Gly and the low kcat/Km values with gamma-L-Glu-L-Cys-beta-Ala (0.03 and 0.01 mM-1s-1 for unactivated and activated enzyme, respectively). Utilization of binding energy from a correctly positioned carboxyl group in the glycine residue (10 and 17 kJ/mol for unactivated and activated enzyme, respectively) therefore also appears to be required for optimal activity and activation. A conformational change in the microsomal glutathione transferase upon treatment with N-ethylmaleimide or trypsin, which allows utilization of binding energy from the alpha-amino group of GSH as well as the glycine carboxyl in catalysis, is suggested to account for at least part of the activation of the enzyme.