Human and murine cytotoxic T lymphocyte serine proteases: subsite mapping with peptide thioester substrates and inhibition of enzyme activity and cytolysis by isocoumarins

The active site structures of human Q31 granzyme A, murine granzymes (A, B, C, D, E, and F), and human granzymes (A, B, and 3) isolated from cytotoxic T lymphocytes (CTL) were studied with peptide thioester substrates, peptide chloromethyl ketone, and isocoumarin inhibitors. Human Q31, murine, and human granzyme A hydrolyzed Arg- or Lys-containing thioesters very efficiently with kcat/KM of 10(4)-10(5) M-1 s-1. Murine granzyme B was found to have Asp-ase activity and hydrolyzed Boc-Ala-Ala-Asp-SBzl with a kcat/KM value of 2.3 X 10(5) M-1 s-1. The rate was accelerated 1.4-fold when the 0.05 M NaCl in the assay was replaced with CaCl2. The preparation of granzyme B also had significant activity toward Boc-Ala-Ala-AA-SBzl substrates, where AA was Asn, Met, or Ser [kcat/KM = (4-5) X 10(4) M-1 s-1]. Murine granzymes C, D, and E did not hydrolyze any thioester substrate but contained minor contaminating activity toward Arg- or Lys-containing thioesters. Murine granzyme F had small activity toward Suc-Phe-Leu-Phe-SBzl, along with some contaminating trypsin-like activity. Human Q31 granzyme A, murine, and human granzyme A were inhibited quite efficiently by mechanism-based isocoumarin inhibitors substituted with basic groups (guanidino or isothiureidopropoxy). Although the general serine protease inhibitor 3,4-dichloroisocoumarin (DCI) inactivated these tryptases poorly, it was the best isocoumarin inhibitor for murine granzyme B (kobs/[I] = 3700-4200 M-1 s-1). Murine and human granzyme B were also inhibited by Boc-Ala-Ala-Asp-CH2Cl; however, the inhibition was less potent than that with DCI. DCI, 3-(3-amino-propoxy)-4-chloroisocoumarin, 4-chloro-3-(3-isothiureidopropoxy)isocoumarin, and 7-amino-4-chloro-3-(3-isothiureidopropoxy)isocoumarin inhibited Q31 cytotoxic T lymphocyte mediated lysis of human JY lymphoblasts (ED50 = 0.5-5.0 microM).     

Peroxidase activity of catalase modified by progesterone

Catalase conjugates with 3, 7, 9 and 42 progesterone molecules were obtained by the reaction between the enzyme and N-oxy-succinimide ether of 3-0-carboxymethyloxime of progesterone. The enzyme modified by 42 progesterone molecules is effective in o-dianisidine oxidation by hydrogen peroxide and has a kcat/KM value of 512 M-1 s-1. The catalase conjugates with 3, 7 and 9 progesterone molecules exhibit a high activity during o-dianisidine oxidation by cumene hydroperoxide. The activity of conjugates is higher than that of the native non-modified enzyme in the same reaction. The maximum effectiveness was observed for catalase modified by 7 progesterone molecules. This conjugate is characterized by kcat/KM of 99,000 M-1 s-1 at 30 degrees C. The effect of the degree of enzyme modification on the kinetic parameters of o-dianisidine oxidation by H2O2 and cumene hydroperoxide is discussed.     

Human complement proteins D, C2, and B. Active site mapping with peptide thioester substrates

The specificity and reactivity of complement serine proteases D, B, Bb, C2, and C2a were determined using a series of peptide thioester substrates. The rates of thioester hydrolysis were measured using assay mixtures containing the thiol reagent 4,4'-dithiodipyridine at pH 7.5. Each substrate contained a P1 arginine residue, and the effect of various groups and amino acids in the P2, P3, P4, and P5 positions was determined using kcat/Km values to compare reactivities. Among peptide thioesters corresponding to the activation site sequence in B, dipeptide thioesters containing a P2 lysine residue were the best substrates for D. Extending the chain to include a P3 or P4 amino acid resulted in loss of activity, and neither the tripeptide nor the tetrapeptide containing the cleavage sequence of B was hydrolyzed. Overall, D cleaved fewer substrates and was 2-3 orders of magnitude less reactive than C1s against some thioester substrates. C2 and fragment C2a had comparable reactivities and hydrolyzed peptides containing Leu-Ala-Arg and Leu-Gly-Arg, which have the same sequence as the cleavage sites of C3 and C5, respectively. The best substrates for C2 and C2a were Z-Gly-Leu-Ala-Arg-SBzl and Z-Leu-Gly-Leu-Ala-Arg-SBzl, respectively, where Bzl is benzyl. B was the least reactive among these complement enzymes. The best substrate for B was Z-Lys-Arg-SBzl with a kcat/Km value of 1370 M-1 s-1. The catalytic fragment of B, Bb, had higher activity toward these peptide thioester substrates. The best substrate for Bb was Z-Gly-Leu-Ala-Arg-SBzl with a kcat/Km similar to C2a and 10 times higher than the value for B. Both C2a and Bb were considerably more reactive against C3-like than C5-like substrates. Bovine trypsin hydrolyzed thioester substrates with kcat/Km approximately 10(3) higher than the complement enzymes. These thioester substrates for D, B, and C2 should be quite useful in kinetic and active site studies of the purified enzymes.     

Interaction between bovine trypsin and a synthetic peptide containing 28 residues of the bait region of human alpha 2-macroglobulin.

The time course of the interaction between trypsin and a synthetic peptide corresponding to a segment (residues 676-703) of the bait region (residues 666-706) of human alpha 2-macroglobulin (alpha 2M) was studied by measuring the generation of cleavage products as a function of time by HPLC. Three primary cleavage sites for trypsin were present in the synthetic peptide. The fastest cleavage occurred at the bond corresponding to Arg696-Leu in alpha 2M with an estimated kcat/Km = 1-2 x 10(6) M-1.s-1. This value is of the same magnitude as that characterizing the interaction of alpha 2M and trypsin when taking into account the fact that alpha 2M is a tetramer, kcat/Km = 5 x 10(6) M-1.s-1 [Christensen, U. & Sottrup-Jensen, L. (1984) Biochemistry 23, 6619-6626]. The values of kcat/Km for cleavage at bonds corresponding to Arg681-Val and Arg692-Gly in alpha 2M were 1.5 x 10(5) M-1.s-1 and 1.3 x 10(5) M-1.s-1, respectively. Cleavage of intermediate product peptides was slower, with kcat/Km in the range 13-1.3 x 10(6) M-1.s-1. The value of Km determined for fast cleavage in the synthetic peptide was 8-10 microM. 1H-NMR spectroscopy indicated no ordered structure of the peptide. Hence, the very fast cleavage of the peptide is compatible with a loose structure that readily adopts a conformation favorable for recognition and cleavage by trypsin.     

The reaction of acetyldithio-CoA, a readily enolized analog of acetyl-CoA with thiolase from Zoogloea ramigera

Acetyldithio-CoA has been shown to be a competent nucleophilic substrate but not an electrophilic substrate for the Claisen condensation catalyzed by thiolase, which normally dimerizes acetyl (Ac)-CoA to acetoacetyl-CoA. Acting as the nucleophile, the kcat/Km for dithioacetyl-CoA is comparable to that of Ac-CoA, the normal substrate. With acetoacetyl-pantetheine acetylating the thiolase to provide the electrophile, the kcat and kcat/Km for the Claisen condensation are 2.1 s-1 and 8.3 X 10(4) M-1 s-1, respectively. The product of the reaction is 3-ketobutyryldithio-CoA. The 3-ketobutyryldithio-CoA has a spectrally determined pKa of 6.55 and the enolate has a lambda max of 357 nm, epsilon 357 = 21,000 cm-1 M-1. Product analysis indicates that acetyldithio-CoA does not serve as the electrophilic partner in the enzymic condensation. This failure is attributed to the inability demonstrated in this study of acetyldithio-CoA to thioacetylate the active site Cys89 of the Zoogloea ramigera thiolase. 1H NMR studies in D2O indicate that thiolase catalyzes the exchange of the alpha-hydrogens, without Cys89 being acetylated, with a rate of 0.63 +/- 0.25 s-1. In the presence of a large excess of acetoacetyl-pantetheine, present to acetylate Cys89 and prevent the thiolytic back reaction, solvent exchange of the alpha-hydrogens can still be detected by observing the isotope-shifted 13C NMR spectrum of [2-13C]acetyldithio-CoA. The exchange of the acetyldithio-CoA alpha-hydrogens with solvent promoted by the acetylated enzyme, must proceed at a rate comparable to that of the condensation reaction.     

A protein engineering study of the role of aspartate 158 in the catalytic mechanism of papain

The controversy concerning the various suggested roles for the side chain of Asp158 in the active site of papain has been clarified by using site-directed mutagenesis. Both wild-type papain and an Asp158 Asn variant were produced in a baculovirus-insect cell expression system, purified to homogeneity from the culture, and characterized kinetically. With CBZ-Phe-Arg-MCA as substrate, the kcat/KM and kcat values obtained for the Asp158Asn papain are 20,000 M-1.s-1 and 34 s-1, respectively, as compared with values of 120,000 M-1.s-1 and 51 s-1 obtained for the wild-type papain. In addition, the pH-(kcat/KM) profile for the Asp158Asn enzyme is shifted relative to that for the wild-type enzyme to lower values by approximately 0.3 pH unit. This shows clearly that Asp158 is not, as previously postulated, an essential catalytic residue. In addition, the pH dependency data are interpreted to indicate that, contrary to earlier suggestions, the negatively charged side chain of Asp158 does not significantly stabilize the active-site thiolate-imidazolium ion pair. However, its presence does influence the pKa's associated with ion-pair formation in a manner compatible with electrostatic considerations.     

Contribution of the glutamine 19 side chain to transition-state stabilization in the oxyanion hole of papain

The existence of an oxyanion hole in cysteine proteases able to stabilize a transition-state complex in a manner analogous to that found with serine proteases has been the object of controversy for many years. In papain, the side chain of Gln19 forms one of the hydrogen-bond donors in the putative oxyanion hole, and its contribution to transition-state stabilization has been evaluated by site-directed mutagenesis. Mutation of Gln19 to Ala caused a decrease in kcat/KM for hydrolysis of CBZ-Phe-Arg-MCA, which is 7700 M-1 s-1 in the mutant enzyme as compared to 464,000 M-1 s-1 in wild-type papain. With a Gln19Ser variant, the activity is even lower, with a kcat/KM value of 760 M-1 s-1. The 60- and 600-fold decreases in kcat/KM correspond to changes in free energy of catalysis of 2.4 and 3.8 kcal/mol for Gln19Ala and Gln19Ser, respectively. In both cases, the decrease in activity is in large part attributable to a decrease in kcat, while KM values are only slightly affected. These results indicate that the oxyanion hole is operational in the papain-catalyzed hydrolysis of CBZ-Phe-Arg-MCA and constitute the first direct evidence of a mechanistic requirement for oxyanion stabilization in the transition state of reactions catalyzed by cysteine proteases. The equilibrium constants Ki for inhibition of the papain mutants by the aldehyde Ac-Phe-Gly-CHO have also been determined. Contrary to the results with the substrate, mutation at position 19 of papain has a very small effect on binding of the inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)     

von Willebrand factor is a cofactor for thrombin-catalyzed cleavage of the factor VIII light chain

The proteolytic activation of highly purified, heterodimeric porcine factor VIII and factor VIII-von Willebrand factor complex by thrombin was compared at I 0.17, pH 7.0, 22 degrees C. During the activation of factor VIII, heavy-chain cleavage is necessary to activate the procoagulant function, whereas light-chain cleavage is required to dissociate factor VIII from von Willebrand factor. The kinetics of activation of free factor VIII and factor VIII-von Willebrand factor complex were identical. The steady-state kinetics of thrombin-catalyzed heavy-chain cleavages and light-chain cleavage of factor VIII either free or in complex with von Willebrand factor were studied using sodium dodecyl sulfate-polyacrylamide gel radioelectrophoresis and scanning densitometry of fragments derived from 125I-labeled factor VIII. Association of factor VIII with von Willebrand factor resulted in an 8-fold increase in the catalytic efficiency (kcat/Km) of light-chain cleavage (from 7 x 10(6) to 54 x 10(6) M-1 s-1). The catalytic efficiencies of heavy-chain cleavage at position 372 (approximately 6 x 10(6) M-1 s-1) and position 740 (approximately 100 x 10(6) M-1 s-1) were not affected by von Willebrand factor. We conclude that von Willebrand factor promotes cleavage of the factor VIII light chain by thrombin which is followed by rapid dissociation of the complex, so that the rate-limiting step becomes heavy-chain cleavage at position 372. This accounts for the observation that von Willebrand factor has no effect on the kinetics of activation of factor VIII by thrombin.     

Aminonaphthalenesulfonamides, a new class of modifiable fluorescent detecting groups and their use in substrates for serine protease enzymes.

A series of new compounds, 6-amino-1-naphthalenesulfonamides (ANSN), were used as fluorescent detecting groups for substrates of amidases. These compounds have a high quantum fluorescent yield, and the sulfonyl moiety permits a large range of chemical modification. Fifteen ANSN substrates with the structure (N alpha-Z)Arg-ANSNR1R2 were synthesized and evaluated for their reactivity with 8 proteases involved in blood coagulation and fibrinolysis. Thrombin, activated protein C, and urokinase rapidly hydrolyzed substrates with monosubstituted sulfonamide moieties (R1 = H). The maximum rate of substrate homologue). The hydrolysis rates for substrates with branched substituents were slower than their linear analogues. Monosubstituted (N alpha-Z)Arg-ANSNR1R2 possessing cyclohexyl or benzyl groups in the sulfonamide moiety were hydrolyzed by these three enzymes at rates similar to that of the n-butyl homologue (except the cyclohexyl compound for u-PA). Factor Xa rapidly hydrolyzed substrates with short alkyl chains, especially when R1 = R2 = CH3 or C2H5. Lys-plasmin and rt-PA demonstrated low activity with these compounds, and the best results were accomplished for monosubstituted compounds when R2 = benzyl (for both enzymes). Factor VIIa and factor IXa beta exhibited no activity with these substrates. A series of 14 peptidyl ANSN substrates were synthesized, and their reactivity for the same 8 enzymes was evaluated. Thrombin, factor Xa, APC, and Lys-plasmin hydrolyzed all of the substrates investigated. Urokinase, rt-PA, and factor IXa beta exhibited reactivity with a more limited group of substrates, and factor VIIa hydrolyzed only one compound (MesD-LGR-ANSN(C2H5)2). The substrate ZGGRR-ANSNH (cyclo-C6H11) showed considerable specificity for APC in comparison with other enzymes (kcat/KM = 19,300 M-1 s-1 for APC, 1560 for factor IIa, and 180 for factor Xa). This kinetic advantage in substrate hydrolysis was utilized to evaluate the activation of protein C by thrombin in a continuous assay format. Substrate (D-LPR-ANSNHC3H7) was used to evaluate factor IX activation by the factor VIIa/tissue factor enzymatic complex in a discontinuous assay. A comparison between the commercially available substrate chromozyme TH (p-nitroanilide) and the ANSN substrate with the same peptide sequence (TosGPR) demonstrated that aminonaphthalenesulfonamide increased the specificity (kcat/KM) of substrate hydrolysis by thrombin more than 30 times, with respect to factor Xa substrate hydrolysis.     

Vampire bat salivary plasminogen activator exhibits a strict and fastidious requirement for polymeric fibrin as its cofactor, unlike human tissue-type plasminogen activator. A kinetic analysis.

The vampire bat salivary plasminogen activator (BatPA) is virtually inactive toward Glu-plasminogen in the absence of a fibrin-like cofactor, unlike human tissue-type plasminogen activator (tPA) (the kcat/Km values were 4 and 470 M-1 s-1, respectively). In the presence of fibrin II, tPA and BatPA activated Glu-plasminogen with comparable catalytic efficiencies (158,000 and 174,000 M-1 s-1, respectively). BatPA's cofactor requirement was partially satisfied by polymeric fibrin I (54,000 M-1 s-1), but monomeric fibrin I was virtually ineffective (970 M-1 s-1). By comparison, a variety of monomeric and polymeric fibrin-like species markedly enhanced tPA-mediated activation of Glu-plasminogen. Fragment X polymer was 2-fold better but 9-fold worse as cofactor for tPA and BatPA, respectively, relative to fibrin II. Fibrinogen, devoid of plasminogen, was a 10-fold better cofactor for tPA than fibrinogen rigorously depleted of plasminogen, Factor XIII, and fibronectin; the enhanced stimulatory effect of the less-purified fibrinogen was apparently due to the presence of Factor XIII. By contrast, the two fibrinogen preparations were equally poor cofactors of BatPA-mediated activation of Glu-plasminogen. BatPA possessed only 23 and 4% of the catalytic efficiencies of tPA and two-chain tPA, respectively, in hydrolyzing the chromogenic substrate Spectrozyme tPA. However in the presence of fibrin II, BatPA and tPA exhibited similar kcat/Km values for the hydrolysis of Spectrozyme tPA. Our data revealed that BatPA, unlike tPA, displayed a strict and fastidious requirement for polymeric fibrin I or II. Consequently, BatPA may preferentially promote plasmin generation during a narrow temporal window of fibrin formation and dissolution.     

Observation of a chloride-dependent intermediate during catalysis by angiotensin converting enzyme using radiationless energy transfer

Stopped-flow radiationless energy-transfer kinetics have been used to examine the effects of chloride on the hydrolysis of Dns-Lys-Phe-Ala-Arg by angiotensin converting enzyme. The kinetic constants for hydrolysis at pH 7.5 and 22 degrees C in the presence of 300 mM sodium chloride were KM = 28 microM and kcat = 110 s-1, and in its absence, KM = 240 microM and kcat = 68 s-1. The apparent binding constant for chloride was 4 mM, and the extent of chloride activation in terms of kcat/KM was 14-fold. The effects of chloride on the pre-steady-state were examined at 2 degrees C. In the presence of chloride, two distinct enzyme-substrate complexes were observed, suggesting multiple steps in substrate binding. The initial complex was formed during the mixing period (kobsd greater than 200 s-1) while the second complex was formed much more slowly (kobsd = 40 s-1 when [S] = 5 microM and [NaCl] = 150 mM). Strikingly, in the absence of chloride, only a single, rapidly formed enzyme-substrate complex was observed. These results are consistent with a nonessential activator kinetic mechanism in which the slow step reflects conversion of an initially formed complex, (E X Cl- X S)1, to a more tightly bound complex, (E X Cl- X S)2.     

Fluorescence-based continuous assay for the aspartyl protease of human immunodeficiency virus-1

5-Dimethylaminoaphthalene-1-sulfonyl-Ser-Gln-Asn-Tyr-Pro-Ile-Val-T rp (Dns-SQNYPIVW) is a fluorescent substrate for the aspartyl protease of human immunodeficiency virus-1. In intact substrate, fluorescence of Trp (lambda ex 290 nm, lambda em 360 nm) was 60% quenched by energy transfer to the dansyl group. Protease-catalyzed cleavage at the Tyr-Pro bond abolished the energy transfer, and the consequent increase in Trp fluorescence was used to follow the enzymatic reaction. At substrate concentrations less than 60 microM, initial reaction velocity increased as a linear function of substrate concentration, with kcat/KM = 9700 M-1 s-1. Limited solubility and internal fluorescence quenching precluded a determination of KM for Dns-SQNYPIVW, but this was clearly greater than 100 microM.     

Characterization of the kinetic pathway for fibrin promotion of alpha-thrombin-catalyzed activation of plasma factor XIII

Kinetic and thermodynamic studies are presented showing that the cofactor activity of fibrin I (polymerized des-A fibrinogen) in the alpha-thrombin-catalyzed proteolysis of activation peptide (AP) from plasma factor XIII can be attributed to formation of a fibrin I-plasma factor XIII complex (Kd = 65 nM), which is processed by alpha-thrombin more efficiently (kcat/Km = 1.2 x 10(7) M-1 s-1) than free, uncomplexed plasma factor XIII (kcat/Km = 1.4 x 10(5) M-1 s-1). The increase in the specificity constant (kcat/Km) is shown to be largely due to an increase in the apparent affinity of alpha-thrombin for the complex of plasma factor XIII and fibrin I, as reflected by the 30-fold decrease in the Michaelis constant observed for fibrin I bound plasma factor XIII relative to that for uncomplexed plasma factor XIII. Analysis of the initial rates of alpha-thrombin-catalyzed hydrolysis of fibrinopeptide B (FPB) from fibrin I polymer in the presence of plasma factor XIII indicated that alpha-thrombin bound to fibrin I in the ternary complex of alpha-thrombin, plasma factor XIII, and fibrin I polymer is competent to catalyze cleavage of both FPB from fibrin I and AP from plasma factor XIII. This observation is consistent with the view that alpha-thrombin within the ternary complex is anchored to fibrin I polymer through a binding site distinct from the active site (an exosite) and that the active site is alternatively complexed with the AP moiety of plasma factor XIII or the FPB moiety of fibrin I. This conclusion is supported by the observation that a 12-residue peptide, which binds to an exosite of alpha-thrombin and blocks the interaction of alpha-thrombin with fibrinogen and fibrin, competitively inhibits alpha-thrombin-catalyzed release of both FPB and AP from the fibrin I-plasma factor XIII complex.     

Enzymatic catalysis of proton transfer at carbon: activation of triosephosphate isomerase by phosphite dianion

More than 80% of the rate acceleration for enzymatic catalysis of the aldose-ketose isomerization of (R)-glyceraldehyde 3-phosphate (GAP) by triosephosphate isomerase (TIM) can be attributed to the phosphodianion group of GAP [Amyes, T. L., O'Donoghue, A. C., and Richard, J. P. (2001) J. Am. Chem. Soc. 123, 11325-11326]. We examine here the necessity of the covalent connection between the phosphodianion and triose sugar portions of the substrate by "carving up" GAP into the minimal neutral two-carbon sugar glycolaldehyde and phosphite dianion pieces. This "two-part substrate" preserves both the alpha-hydroxycarbonyl and oxydianion portions of GAP. TIM catalyzes proton transfer from glycolaldehyde in D2O, resulting in deuterium incorporation that can be monitored by 1H NMR spectroscopy, with kcat/Km = 0.26 M-1 s-1. Exogenous phosphite dianion results in a very large increase in the observed second-order rate constant (kcat/Km)obsd for turnover of glycolaldehyde, and the dependence of (kcat/Km)obsd on [HPO32-] exhibits saturation. The data give kcat/Km = 185 M-1 s-1 for turnover of glycolaldehyde by TIM that is saturated with phosphite dianion so that the separate binding of phosphite dianion to TIM results in a 700-fold acceleration of proton transfer from carbon. The binding of phosphite dianion to the free enzyme (Kd = 38 mM) is 700-fold weaker than its binding to the fleeting complex of TIM with the altered substrate in the transition state (Kd = 53 muM); the total intrinsic binding energy of phosphite dianion in the transition state is 5.8 kcal/mol. We propose a physical model for catalysis by TIM in which the intrinsic binding energy of the substrate phosphodianion group is utilized to drive closing of the "mobile loop" and a protein conformational change that leads to formation of an active site environment that is optimally organized for stabilization of the transition state for proton transfer from alpha-carbonyl carbon.     

Kinetic isotope effect and the presteady-state kinetics of the reaction catalyzed by the bacterial formate dehydrogenase

The primary kinetic isotope effect of the reaction catalyzed by NAD+-dependent formate dehydrogenase (EC 1.2.1.2.) from the methylotrophic bacterium Pseudomonas sp. 101 has been studied. Analysis of the ratios HVm/DVm and H(Vm/KM)/D(Vm/KM) in the pH range 6.1-7.9 showed that the transfer of hydride ion in ternary enzyme-substrate complex is a limiting step of the reaction, and the formate binding to the binary complex (formate dehydrogenase + NAD+) reached equilibrium when the pH of the medium was increased. An approach has been developed to determine the elementary constants of substrate association (kon) and dissociation (koff) at the stages of the binary--ternary enzyme-substrate complexes for the random equilibrium 2-substrate kinetic mechanism. The kon and koff values obtained for the bacterial formate dehydrogenase by using the proposed approach for NAD+ were (4.8 +/- 0.8)*10(5)M-1s-1 and (90 +/- 10) s-1, and for formate (2.0 +/- 1.0)*10(4) M-1s-1 and (60 +/- 20) s-1, respectively.     

Rhinovirus 3C protease catalyzes efficient cleavage of a fluorescein-labeled peptide affording a rapid and robust assay.

The 3C protease encoded by human rhinovirus type 2 catalyzes with equal efficiency cleavage of a peptide substrate with or without a fluorescein label attached to the amino acid at the P7' position. Substrates Ac-MEALFQGPLQYKDL-NH2 and MEALFQGPLQYKE(fluorescein)L are hydrolyzed with values of Vmax/KM of 970 M-1 s-1 and 1100 M-1 s-1, respectively. With the labeled substrate, HPLC achieves separation of substrate and product in 2.5 min. Separation in as little as 12 s is feasible. Fluorescein was derivatized so that it could be incorporated into peptides using automated solid-phase peptide synthesis.     

Mapping the active site of meprin-A with peptide substrates and inhibitors

The extended substrate-binding site of meprin-A, a tetrameric metalloendopeptidase from brush border membranes of mouse kidney proximal tubules, was mapped with a series of peptide substrates. Previous studies led to the development of the chromogenic substrate Phe5(4-nitro)bradykinin for meprin-A. With this substrate, several biologically active peptides were screened as alternate substrate inhibitors, and, of these, bradykinin (RPPGFSPFR) was found to be the best substrate with a single cleavage site (Phe5-Ser6). Three types of bradykinin analogues were used for a systematic investigation of substrate specificity: (1) nonchromogenic bradykinin analogues with substitutions in the P3 to P3' subsites were used as alternative substrate inhibitors of nitrobradykinin hydrolysis, (2) analogues of nitrobradykinin with variations in the P1' position were tested as substrates, and (3) intramolecularly quenched fluorogenic bradykinin analogues with substitutions in the P1 to P3 sites were tested as substrates. A wide variety of substitutions in P1' had little effect on KM (174-339 microM) but markedly affected kcat (51.5 s-1 = A greater than S greater than R greater than F greater than K greater than T greater than E = 0). Substitutions in P1 had a greater effect on KM (366 microM-2.46 mM) and also strongly affected kcat (98.5 s-1 = A greater than F much greater than L greater than E greater than K = 2.4 s-1). The variety of allowed cleavages indicates that meprin-A does not have strict requirements for residues adjacent to the cleavage site. Substitutions farther from the scissle bond also affected binding and hydrolysis, demonstrating that multiple subsite interactions are involved in meprin-A action.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reactions of prostaglandin H synthase in the presence of the stabilizing agents diethyldithiocarbamate and glycerol

Both cyclooxygenase and peroxidase reactions of prostaglandin H synthase were studied in the presence and absence of diethyldithiocarbamate and glycerol at 4 degrees C in phosphate buffer (pH 8.0). Diethyldithiocarbamate reacts with the high oxidation state intermediates of prostaglandin H synthase; it protects the enzyme from bleaching and loss of activity by its ability to act as a reducing agent. For the reaction of diethyldithiocarbamate with compound I, the second-order rate constant k2,app, was found to fall within the range of 5.8 x 10(6) +/- 0.4 x 10(6) M-1.s-1 less than k2,app less than 1.8 x 10(7) +/- 0.1 x 10(7) M-1.s-1. The reaction of diethyldithiocarbamate with compound II showed saturation behavior suggesting enzyme-substrate complex formation, with kcat = 22 +/- 3 s-1, Km = 67 +/- 10 microM, and the second-order rate constant k3,app = 2.0 x 10(5) +/- 0.2 x 10(5) M-1.s-1. In the presence of both diethyldithiocarbamate and 30% glycerol, the parameters for compound II are kcat = 8.8 +/- 0.5 s-1, Km = 49 +/- 7 microM, and k3,app = 1.03 x 10(5) +/- 0.07 x 10(5) M-1.s-1. The spontaneous decay rate constants of compounds I and II (in the absence of diethyldithiocarbamate) are 83 +/- 5 and 0.52 +/- 0.05 s-1, respectively, in the absence of glycerol; in the presence of 30% glycerol they are 78 +/- 5 and 0.33 +/- 0.02 s-1, respectively. Neither cyclooxygenase activity nor the rate constant for compound I formation using 5-phenyl-4-pentenyl-1-hydroperoxide is altered by the presence of diethyldithiocarbamate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic investigations of the reactions of cytochrome c oxidase with hydrogen peroxide

The reaction of H2O2 with reduced cytochrome c oxidase was investigated with rapid-scan/stopped-flow techniques. The results show that the oxidation rate of cytochrome a3 was dependent upon the peroxide concentration (k = 2 X 10(4) M-1 X s-1). Cytochrome a and CuA were oxidised with a maximal rate of approx. 20 s-1, indicating that the rate of internal electron transfer was much slower with H2O2 as the electron acceptor than with O2 (k greater than or equal to 700 s-1). Although other explanations are possible, this result strongly suggests that in the catalytic cycle with oxygen as a substrate the internal electron-transfer rate is enhanced by the formation of a peroxo-intermediate at the cytochrome a3-CuB site. It is shown that H2O2 took up two electrons per molecule. The reaction of H2O2 with oxidised cytochrome c oxidase was also studied. It is shown that pulsed oxidase readily reacted with H2O2 (k approximately 700 M-1 X s-1). Peroxide binding is followed by an H2O2-independent conformational change (k = 0.9 s-1). Resting oxidase partially bound H2O2 with a rate similar to that of pulsed oxidase; after H2O2 binding the resting enzyme was converted into the pulsed conformation in a peroxide-independent step (k = 0.2 s-1). Within 5 min, 55% of the resting enzyme reacted in a slower process. We conclude from the results that oxygenated cytochrome c oxidase probably is an enzyme-peroxide complex.     

Clostridium histolyticum collagenase: development of new thio ester, fluorogenic, and depsipeptide substrates and new inhibitors

A new series of thio ester, depsipeptide, and peptide substrates have been synthesized for the bacterial enzyme Clostridium histolyticum collagenase. The hydrolysis of the depsipeptide substrate was followed on a pH stat, and thio ester hydrolysis was measured by inclusion of the chromogenic thiol reagent 4,4'-dithiopyridine in the assay mixture. The best thio ester substrate, Boc-Abz-Gly-Pro-Leu-SCH2CO-Pro-Nba, had a kcat/KM of 63 000 M-1 s-1, while several shorter thio ester sequences were inactive as substrates. In general, the peptide analogues of all the reactive thio ester substrates were shown to be hydrolyzed 5-10 times faster by collagenase. In one case (Z-Gly-Pro-Leu-Gly-Pro-NH2) where a comparison was made, the peptide substrate was respectively 8- and 106-fold more readily hydrolyzed than the corresponding thio ester and ester substrates. Cleavages of the two fluorescence-quench substrates Abz-Gly-Pro-Leu-Gly-Pro-Nba and Abz-Gly-Pro-Leu-SCH2CO-Pro-Nba could be easily followed fluorogenically since a 5-10-fold increase in fluorescence occurred upon hydrolysis. The fluorescent peptide substrate is the best synthetic substrate known for C. histolyticum collagenase with a kcat/KM value of 490 000 M-1 s-1. A series of new reversible inhibitors were developed by the attachment of zinc ligating groups (hydroxamic acid, carboxymethyl, and thiol) to various peptide sequences specific for C. histolyticum collagenase. The shorter peptides designed to bind to either the P3-P1 or P1'-P3' subsites were poor to moderate inhibitors. The thiol HSCH2CH2CO-Pro-Nba had the lowest K1 (0.02 mM).(ABSTRACT TRUNCATED AT 250 WORDS)