Reaction of ferrous lactoperoxidase with hydrogen peroxide and dioxygen: an anaerobic stopped-flow study

Lactoperoxidase (LPO) is found in mucosal surfaces and exocrine secretions including milk, tears, and saliva and has physiological significance in antimicrobial defense which involves (pseudo-)halide oxidation. LPO compound III (a ferrous-dioxygen complex) is known to be formed rapidly by an excess of hydrogen peroxide and could participate in the observed catalase-like activity of LPO. The present anaerobic stopped-flow kinetic analysis was performed in order to elucidate the catalytic mechanism of LPO and the kinetics of compound III formation by probing the reactivity of ferrous LPO with hydrogen peroxide and molecular oxygen. It is shown that ferrous LPO heterolytically cleaves hydrogen peroxide forming water and oxyferryl LPO (compound II). The two-electron oxidation reaction follows second-order kinetics with the apparent bimolecular rate constant being (7.2+/-0.3) x 10(4) M(-1) s(-1) at pH 7.0 and 25 degrees C. The H2O2-mediated conversion of compound II to compound III follows also second-order kinetics (220 M(-1) s(-1) at pH 7.0 and 25 degrees C). Alternatively, compound III is also formed by dioxygen binding to ferrous LPO at an apparent bimolecular rate constant of (1.8+/-0.2) x 10(5) M(-1) s(-1). Dioxygen binding is reversible and at pH 7.0 the dissociation constant (K(D)) of the oxyferrous form is 6 microM. The rate constant of dioxygen dissociation from compound III is higher than conversion of compound III to ferric LPO, which is not affected by the oxygen concentration and follows a biphasic kinetics. A reaction cycle including the redox intermediates compound II, compound III, and ferrous LPO is proposed, which explains the observed (pseudo-)catalase activity of LPO in the absence of one-electron donors. The relevance of these findings in LPO catalysis is discussed.     

Reaction of myeloperoxidase with its product HOCl.

The reaction of human myeloperoxidase with its product, hypochlorous acid was investigated using both rapid-scan spectrophotometry and the stopped-flow technique. In the reaction of myeloperoxidase with hypochlorous acid a primary compound is found with properties similar to that of compound I and which is converted into compound II. The primary reaction is strongly pH-dependent. At pH 7.2 the reaction is too fast to be measured but at higher pH values it is possible to determine the apparent second-order rate constant. Its value decreases to about 2 x 10(7) M-1.s-1 at pH 8.3 and to 2.3 (+/- 0.4) x 10(6) M-1.s-1 at pH 9.2, respectively. The dissociation constant for the formation of the primary compound is 25.7 (+/- 15.3) microM at pH 9.2 and about 2.5 microM at pH 8.3. The apparent second-order rate constant for the formation of compound II is hardly affected by pH and varies between 2 to 5 x 10(4) M-1.s-1 at pH 10.2 and pH 8.3, respectively. Reaction of myeloperoxidase with hypochlorous acid also resulted in irreversible partial bleaching of the chromophore. Chloride, which is a substrate of the enzyme not only protects myeloperoxidase against bleaching by hypochlorous acid but also competitively inhibits the binding of hypochlorous acid to myeloperoxidase, a process which also has been observed in the reaction with hydrogen peroxide. It is concluded that hypochlorous acid binds at the heme iron to form compound I.     

Kinetics of oxygen binding to ferrous myeloperoxidase

Myeloperoxidase (MPO), which is involved in host defence and inflammation, is a unique peroxidase in having a globin-like standard reduction potential of the ferric/ferrous couple. Intravacuolar and exogenous MPO released from stimulated neutrophils has been shown to exist in the oxyferrous form, called compound III. To investigate the reactivity of ferrous MPO with molecular oxygen, a stopped-flow kinetic analysis was performed. In the absence of dioxygen, ferrous MPO decays to ferric MPO (0.04 s(-1) at pH 8 versus 1.4 s(-1) at pH 5). At pH 7.0 and 25 degrees C, compound III formation (i.e., binding of dioxygen to ferrous MPO) occurs with a rate constant of (1.1+/-0.1) x 10(4)M(-1)s(-1). The rate doubles at pH 5.0 and oxygen binding is reversible. At pH 7.0, the dissociation equilibrium constant of the oxyferrous form is (173+/-12)microM. The rate constant of dioxygen dissociation from compound III is much higher than conversion of compound III to ferric MPO (which is not affected by the oxygen concentration). This allows an efficient transition of compound III to redox intermediates which actually participate in the peroxidase or halogenation cycle of MPO.     

Kinetics of binding of chicken cystatin to papain

The kinetics of binding of chicken cystatin to papain were studied by stopped-flow fluorometry under pseudo-first-order conditions, i.e., with an excess of inhibitor. All reactions showed first-order behavior, and the observed pseudo-first-order rate constant increased linearly with the cystatin concentration up to the highest concentration that could be studied, 35 microM. The analyses thus provided no evidence for a limiting rate resulting from a conformational change stabilizing an initial encounter complex, in contrast with previous studies of reactions between serine proteinases and their protein inhibitors. The second-order association rate constant for complex formation was 9.9 X 10(6) M-1 s-1 at 25 degrees C, pH 7.4, I = 0.15, for both forms of cystatin, 1 and 2. This value approaches that expected for a diffusion-controlled rate. The temperature dependence of the association rate constant gave an enthalpy of activation at 25 degrees C of 31.5 kJ mol-1 and an entropy of activation at 25 degrees C of -7 J K-1 mol-1, compatible with no appreciable conformational change during the reaction. The association rate constant was independent of pH between pH 6 and 8 but decreased at lower and higher pH in a manner consistent with involvement of an unprotonated acid group with a pKa of 4-4.5 and a protonated basic group with a pKa of 9-9.5 in the interaction. The association rate constant was unaffected by ionic strengths between 0.15 and 1.0 but decreased somewhat at lower ionic strengths. Incubation of the complex between cystatin 2 and papain with an excess of cystatin 1 resulted in slow displacement of cystatin 2 from the complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

The reduction of putidaredoxin reductase by reduced pyridine nucleotides

Putidaredoxin reductase (PdR), an FAD-containing protein, mediates the transfer of electrons from NADH to putidaredoxin in the cytochrome P-450cam-dependent oxidation of camphor. Using stopped-flow spectrophotometry, reduction of putidaredoxin reductase by NADH (70 microM) at 4 degrees C appeared to be a pseudo-first-order process with a rate constant in excess of 600 s-1. The reduction of putidaredoxin reductase by NADPH was much slower with a second-order rate constant of 530 s-1 M-1 at 4 degrees C. The reduction of the enzyme was monitored at several wavelengths: 455 nm to follow flavin reduction; 700 nm to follow the appearance of the long-wavelength charge-transfer complex; and 513 nm to detect the presence of a semiquinone form of the flavoprotein. There was no apparent semiquinone formation observed during reduction. The charge-transfer complex can be formed in the presence of NAD+, whereas, no charge-transfer band could be detected when PdR was reduced with NADPH. The titration of chemically or NADPH-reduced putidaredoxin reductase with either a stoichiometric or an excess amount of NAD+ resulted in the formation of a charge-transfer complex, indicating that the reduced form of PdR has a high affinity for NAD+ regardless of the method of reduction. The data presented indicate that putidaredoxin reductase is reduced without the formation of semiquinone intermediate and, upon reduction, forms a tight complex with NAD+. The Keq for the reduction of PdR by NADPH is 1.1 and the midpoint potential for this reaction is -317 +/- 5 mV.     

Kinetics of the reaction of prostaglandin H synthase compound II with ascorbic acid

The reduction of prostaglandin H synthase compound II by ascorbic acid in the presence of diethyldithiocarbamate was studied in 0.1 M phosphate buffer (pH 8.0) at 4.0 +/- 0.5 degrees C, by rapid scan spectrometry and transient state kinetics. A saturation effect and nonzero intercept were observed in the plot of pseudo-first-order rate constant versus ascorbic acid concentration. The saturation behavior suggests formation of a complex between prostaglandin H synthase compound II and ascorbic acid, whereas the nonzero intercept is attributable to the reaction of compound II of prostaglandin H synthase with diethyldithiocarbamate present in the system as a stabilizing agent. A rate equation has been derived which includes all pathways for the conversion of prostaglandin H synthase compound II back to native enzyme. Kinetic parameters for the reduction of compound II by ascorbic acid were obtained. They are the second-order rate constant of (1.4 +/- 0.5) X 10(5) M-1, S-1, for the formation of the compound II-ascorbic acid complex, the first-order rate constant of (14 +/- 4) S-1 for the oxidation-reduction reaction of the complex and its dissociation, and a parameter, Km of 92 +/- 10 microM analogous to the Michaelis-Menten constant. Thus we demonstrate that a quantitative kinetic study on the prostaglandin H synthase reactions can be performed in the presence of diethyldithiocarbamate.     

The reaction of Pseudomonas aeruginosa cytochrome c oxidase with carbon monoxide.

The binding of CO to ascorbate-reduced Pseudomonas cytochrome oxidase was investigated by static-titration, stopped-flow and flash-photolytic techniques. Static-titration data indicated that the binding process was non-stoicheiometric, with a Hill number of 1.44. Stopped-flow kinetics obtained on the binding of CO to reduced Pseudomonas cytochrome oxidase were biphasic in form; the faster rate exhibited a linear dependence on CO concentration with a second-order rate constant of 2 X 10(4) M-1-s-1, whereas the slower reaction rapidly reached a pseudo-first-order rate limit at approx. 1s-1. The relative proportions of the two phases observed in stopped-flow experiments also showed a dependency on CO concentration, the slower phase increasing as the CO concentration decreased. The kinetics of CO recombination after flash-photolytic dissociation of the reduced Pseudomonas cytochrome oxidase-CO complex were also biphasic in character, both phases showing a linear pseudo-first-order rate dependence on CO concentration. The second-order rate constants were determined as 3.6 X 10(4)M-1-s-1 and 1.6 X 10(4)M-1-s-1 respectively. Again the relative proportions of the two phases varied with CO concentration, the slower phase predominating at low CO concentrations. CO dissociation from the enzyme-CO complex measured in the presence of O2 and NO indicated the presence of two rates, of the order of 0.03s-1 and 0.15s-1. When sodium dithionite was used as a reducing agent for the Pseudomonas cytochrome oxidase, the CO-combination kinetics observed by both stopped flow and flash photolysis were extremely complex and not able to be simply analysed.     

Reactions of electron-transfer flavoprotein and electron-transfer flavoprotein: ubiquinone oxidoreductase.

Electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF-Q oxidoreductase) catalyses the re-oxidation of reduced electron-transfer flavoprotein (ETF) with ubiquinone-1 (Q-1) as the electron acceptor. A kinetic assay for the enzyme was devised in which glutaryl-CoA in the presence of glutaryl-CoA dehydrogenase was used to reduce ETFox. and the reduction of Q-1 was monitored at 275 nm. The partial reactions involved in the overall assay system were examined. Glutaryl-CoA dehydrogenase catalyses the rapid reduction of ETFox. to the anionic semiquinone (ETF.-), but reduces ETF.- to the fully reduced form (ETFhq) at a rate that is about 6-fold lower. ETF.-, but not ETFhq, is directly re-oxidized by Q-1 at a rate that, depending on the steady-state concentration of ETF.-, may contribute significantly to the overall reaction. ETF-Q oxidoreductase catalyses rapid disproportionation of ETF.- with an equilibrium constant of about 1.0 at pH 7.8. In the presence of Q-1 it also catalyses the re-oxidation of ETFhq at a rate that is faster than that of the overall reaction. Rapid-scan experiments indicated the formation of ETF.-, but its fractional concentration in the early stages of the re-oxidation of ETFhq is low. The data indicate that the re-oxidation of ETFhq proceeds at a rate that is adequate to account for the overall rate of electron transfer from glutaryl-CoA to Q-1. An unusual property of ETF-Q oxidoreductase seems to be that it not only catalyses the re-oxidation of the reduced forms of ETF but also facilitates the complete reduction of ETFox. to ETFhq by disproportionation of the radical.     

Equilibrium and kinetics of the allosteric transition of GroEL studied by solution X-ray scattering and fluorescence spectroscopy

We have studied the ATP-induced allosteric structural transition of GroEL using small angle X-ray scattering and fluorescence spectroscopy in combination with a stopped-flow technique. With X-ray scattering one can clearly distinguish the three allosteric states of GroEL, and the kinetics of the transition of GroEL induced by 85 microM ATP have been observed directly by stopped-flow X-ray scattering for the first time. The rate constant has been found to be 3-5s(-1) at 5 degrees C, indicating that this process corresponds to the second phase of the ATP-induced kinetics of tryptophan-inserted GroEL measured by stopped-flow fluorescence. Based on the ATP concentration dependence of the fluorescence kinetics, we conclude that the first phase represents bimolecular non-cooperative binding of ATP to GroEL with a bimolecular rate constant of 5.8 x 10(5)M(-1)s(-1) at 25 degrees C. Considering the electrostatic repulsion between negatively charged GroEL (-18 of the net charge per monomer at pH 7.5) and ATP, the rate constant is consistent with a diffusion-controlled bimolecular process. The ATP-induced fluorescence kinetics (the first and second phases) at various ATP concentrations (< 400 microM) occur before ATP hydrolysis by GroEL takes place and are well explained by a kinetic allosteric model, which is a combination of the conventional transition state theory and the Monod-Wyman-Changeux model, and we have successfully evaluated the equilibrium and kinetic parameters of the allosteric transition, including the binding constant of ATP in the transition state of GroEL.     

Nucleotide sequence analysis, overexpression in Escherichia coli and kinetic characterization of Anacystis nidulans catalase-peroxidase

Bifunctional catalase-peroxidases are the least understood type of peroxidases. A high-level expression in Escherichia coli of a fully active recombinant form of a catalase-peroxidase (KatG) from the cyanobacterium Anacystis nidulans (Synechococcus PCC 6301) is reported. Since both physical and kinetic characterization revealed its identity with the wild-type protein, the large quantities of recombinant KatG allowed the examination of both the spectral characteristics and the reactivity of its redox intermediates by using the multi-mixing stopped-flow technique. The homodimeric acidic protein (pI = 4.6) contained high catalase activity (apparent K(m) = 4.8 mM and apparent k(cat) = 8850 s(-1)). Cyanide is shown to be an effective inhibitor of the catalase reaction. The second-order rate constant for cyanide binding to the ferric protein is (6.9 +/- 0.2) x 10(5) M(-1 )s(-1) at pH 7.0 and 15 degrees C and the dissociation constant of the cyanide complex is 17 microM. Because of the overwhelming catalase activity, peroxoacetic acid has been used for compound I formation. The apparent second-order rate constant for formation of compound I from the ferric enzyme and peroxoacetic acid is (1.3 +/- 0.3) x 10(4 )M(-1 )s(-1) at pH 7.0 and 15 degrees C. The spectrum of compound I is characterized by about 40% hypochromicity, a Soret region at 406 nm, and isosbestic points between the native enzyme and compound I at 355 and 428 nm. Rate constants for reduction of KatG compound I by o-dianisidine, pyrogallol, aniline and isoniazid are shown to be (7.3 +/- 0.4) x 10(6) M(-1 )s(-1), (5.4 +/- 0.3) x 10(5) M(-1 )s(-1), (1.6 +/- 0.3) x 10(5) M(-1 )s(-1) and (4.3 +/- 0.2) x 10(4) M(-1 )s(-1), respectively. The redox intermediate formed upon reduction of compound I did not exhibit the classical red-shifted peroxidase compound II spectrum which characterizes the presence of a ferryl oxygen species. Its spectral features indicate that the single oxidizing equivalent in KatG compound II is contained on an amino acid which is not electronically coupled to the heme.     

Stopped-flow and steady-state study of the diphenolase activity of mushroom tyrosinase

The reaction of mushroom (Agaricus bisporus) tyrosinase with dioxygen in the presence of several o-diphenolic substrates has been studied by steady-state and transient-phase kinetics in order to elucidate the rate-limiting step and to provide new insights into the mechanism of oxidation of these substrates. A kinetic analysis has allowed for the first time the determination of individual rate constants for several of the partial reactions that comprise the catalytic cycle. Mushroom tyrosinase rapidly reacts with dioxygen with a second-order rate constant k(+8) = 2.3 x 10(7) M(-)(1) s(-)(1), which is similar to that reported for hemocyanins [(1.3 x 10(6))-(5.7 x 10(7)) M(-)(1) s(-)(1)]. Deoxytyrosinase binds dioxygen reversibly at the binuclear Cu(I) site with a dissociation constant K(D)(O)()2 = 46.6 microM, which is similar to the value (K(D)(O)()2 = 90 microM) reported for the binding of dioxygen to Octopus vulgaris deoxyhemocyanin [Salvato et al. (1998) Biochemistry 37, 14065-14077]. Transient and steady-state kinetics showed that o-diphenols such as 4-tert-butylcatechol react significantly faster with mettyrosinase (k(+2) = 9.02 x 10(6) M(-)(1) s(-)(1)) than with oxytyrosinase (k(+6) = 5.4 x 10(5) M(-)(1) s(-)(1)). This difference is interpreted in terms of differential steric and polar effects that modulate the access of o-diphenols to the active site for these two forms of the enzyme. The values of k(cat) for several o-diphenols are also consistent with steric and polar factors controlling the mobility, orientation, and thence the reactivity of substrates at the active site of tyrosinase.     

Interactions of soluble penicillin-binding protein 2a of methicillin-resistant Staphylococcus aureus with moenomycin.

Kinetics studies in homogeneous aqueous solution showed that solubilized penicillin-binding protein 2a (sPBP2a) of methicillin-resistant Staphylococcus aureus (a bacterial DD-peptidase) was inhibited by the amphiphilic glycolipid antibiotic moenomycin. Inhibition at the peptidase site was determined by competition experiments between moenomycin and the chromophoric beta-lactam nitrocefin. Under conditions of high salt concentration (1 M NaCl), pseudo-first-order rate constants for the reaction of moenomycin with sPBP2a leading to inhibition of acylation by nitrocefin varied with moenomycin concentration in a biphasic fashion. At low moenomycin concentration (<20 microM) little inhibition occurred, but at higher concentrations a linear increase in rate constant with moenomycin concentration was observed, yielding a second-order rate constant of inhibition of 120 s(-)(1) M(-)(1). Since the cmc of moenomycin under these conditions was shown to be ca. 20 microM, the inhibition was concluded to arise from reaction of sPBP2a with a moenomycin micelle. Protein fluorescence studies showed a pseudo-first-order decrease in fluorescence on reaction of the protein with moenomycin. The variation of this rate constant with moenomycin concentration was consistent with reaction of a moenomycin monomer with the protein with a second-order rate constant of 650 s(-)(1) M(-)(1). This monomer reaction did not occur at the DD-peptidase site since its rate was unaffected by prior acylation of the enzyme by benzylpenicillin; nor did it inhibit reaction at that site by beta-lactams. Under low salt conditions (0.175 M NaCl) where reaction could be studied over a greater range of monomer concentrations since the cmc was ca. 120 microM, similar reactions were involved. Under these circumstances, inhibition was concerted with the reaction of moenomycin monomers, although fast premicellar aggregation of moenomycin with the protein also occurred. All moenomycin interactions with sPBP2a were reversible, as revealed by detergent-extraction chromatography. Lower limits to moenomycin off-rates and equilibrium dissociation constants were 7.7 x 10(-)(4) s(-)(1) and 1.2 microM, respectively. Other amphiphiles did not react in exactly the same manner as moenomycin, indicating some degree of specificity in reactions of the latter. sPBP2a did not have detectable affinity for lipid surfaces (Triton X-114 and phosphatidylglycerol vesicles). A general scheme for reaction of moenomycin with sPBP2a is proposed.     

The reaction of Pseudomonas aeruginosa cytochrome c-551 oxidase with oxygen.

The reaction of ascorbate-reduced Pseudomonas cytochrome oxidase with oxygen was studied by using stopped-flow techniques at pH 7.0 and 25 degrees C. The observed time courses were complex, the reaction consisting of three phases. Of these, only the fastest process, with a second-order rate constant of 3.3 X 10(4) M-1.S-1, was dependent on oxygen concentration. The two slower processes were first-order reactions with rates of 1.0 +/- 0.4s-1 and 0.1 +/- 0.03s-1. A kinetic titration experiment revealed that the enzyme had a relatively low affinity constant for oxygen, approx. 10(4)M-1. Kinetic difference spectra were determined for all three reaction phases, showing each to have different characteristics. The fast-phase difference spectrum showed that changes occurred at both the haem c and haem d1 components of the enzyme during this process. These changes were consistent with the haem c becoming oxidized, but with the haem d1 assuming a form that did not correspond to the normal oxidized state, a situation that was not restored even after the second kinetic phase, which reflected further changes in the haem d1 component. The results are discussed in terms of a kinetic scheme.     

Carboxymethylation of an active site glutamic acid residue of ribonuclease F1 iodoacetate

Ribonuclease (RNase) F1 was inactivated by incubation with an excess amount of iodoacetate at pH 5.5, 37 degrees C according to pseudo first-order kinetics. It was protected to various degrees, from inactivation by nucleotides, among which guanosine 2'-phosphate was most effective. The pseudo first-order rate constant was proportional to the reagent concentration, indicating that the reaction in reality follows second-order kinetics. The second-order rate constant was determined to be 25 x 10(-4) M-1 s-1. The inactivation rate was maximal at pH 5.5-6.0. When iodo[2-14C]acetate was used as the reagent, the stoichiometry of incorporation was determined to be 1.1 mol carboxymethyl group per mol of RNase F1 and glutamic acid residue 58 was assigned as the site of modification.     

Dioxygen reactivity and heme redox potential of truncated human cystathionine beta-synthase

Cystathionine beta-synthase (CBS) catalyzes the condensation of serine and homocysteine to cystathionine, which represents the committing step in the transsulfuration pathway. CBS is unique in being a pyridoxal phosphate-dependent enzyme that has a heme cofactor. The activity of CBS under in vitro conditions is responsive to the redox state of the heme, which is distant from the active site and has been postulated to play a regulatory role. The heme in CBS is unusual; it is six-coordinate, low spin, and contains cysteine and histidine as axial ligands. In this study, we have assessed the redox behavior of a human CBS dimeric variant lacking the C-terminal regulatory domain. Potentiometric redox titrations showed a reversible response with a reduction potential of -291 +/- 5 mV versus the normal hydrogen electrode, at pH 7.2. Stopped-flow kinetic determinations demonstrated that Fe(II)CBS reacted with dioxygen yielding Fe(III)CBS without detectable formation of an intermediate species. A linear dependence of the apparent rate constant of Fe(II)CBS decay on dioxygen concentration was observed and yielded a second-order rate constant of (1.11 +/- 0.07) x 10 (5) M (-1) s (-1) at pH 7.4 and 25 degrees C for the direct reaction of Fe(II)CBS with dioxygen. A similar reactivity was observed for full-length CBS. Heme oxidation led to superoxide radical generation, which was detected by the superoxide dismutase (SOD)-inhibitable oxidation of epinephrine. Our results show that CBS may represent a previously unrecognized source of cytosolic superoxide radical.     

The reaction of Aspergillus niger catalase with methyl hydroperoxide.

The formation of Compound I from Aspergillus niger catalase and methyl hydroperoxide (CH3OOH) has been investigated kinetically by means of rapid-scanning stopped-flow techniques. The spectral changes during the reaction showed distinct isobestic points. The second-order rate constant and the activation energy for the formation of Compound I were 6.4 x 10(3) M-1s-1 and 10.4 kcal.mol-1, respectively. After formation of Compound I, the absorbance at the Soret peak returned slowly to the level of ferric enzyme with a first-order rate constant of 1.7 x 10(-3) s-1. Spectrophotometric titration of the enzyme with CH3OOH indicates that 4 mol of peroxide react with 1 mol of enzyme to form 1 mol of Compound I. The amount of Compound I formed was proportional to the specific activity of the catalase. The irreversible inhibition of catalase by 3-amino-1,2,4-triazole (AT) was observed in the presence of CH3OOH or H2O2. The second-order rate constant of the catalase-AT formation in CH3OOH was 3.0 M-1 min-1 at 37 degrees C and pH 6.8 and the pKa value was estimated to be 6.10 from the pH profile of the rate constant of the AT-inhibition. These results indicate that A. niger catalase forms Compound I with the same properties as other catalases and peroxidases, but the velocity of the Compound I formation is lower than that of the others.     

Reaction pathway for inhibition of blood coagulation factor Xa by tick anticoagulant peptide.

The reaction pathway for inhibition of human factor Xa (fXa) by recombinant tick anticoagulant peptide (rTAP) was studied by stopped-flow fluorometry. In the presence of the fluorogenic substrate N-tert-butyloxycarbonyl-L-isoleucyl-L-glutamylglycyl-L-arginyl-7-amido-4 - methylcoumarin (B-IEGR-AMC) and under pseudo-first-order conditions, inhibition appears to occur via a two-step process. Initially, a weak enzyme-inhibitor complex forms with a dissociation constant (Ki) of 68 +/- 6 microM. The initial complex then rearranges to a more stable fXa-rTAP complex with a rate constant (k2) of 123 +/- 5 s-1. The apparent second-order rate constant (k2/Ki) describing formation of the stable complex is (1.8 +/- 0.2) x 10(6) M-1 s-1. Studies of the reaction of rTAP with fXa in the presence of the fluorescent active-site probe p-amino-benzamidine (P) revealed a reaction pathway wherein rTAP initially binds to the fXa-P complex in a two-step process prior to displacing P from the active site. These results indicate that rTAP can bind fXa via a site distinct from the active site (an exosite). The subsequent displacement of P from the active site of fXa by rTAP exhibits a dependence on the concentration of P, indicating that rTAP is locked into the active site in a third step.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of photosynthetic electron transfer in artificial vesicles reconstituted with purified complexes from Rhodobacter capsulatus. I. The interaction of cytochrome c2 with the reaction center

1. The kinetics of the interaction of cytochrome c2 and photosynthetic reaction centers purified from Rhodobacter capsulatus were studied in proteoliposomes reconstituted with a mixture of phospholipids simulating the native membrane (i.e. containing 25% L-alpha-phosphatidylglycerol). 2. At low ionic strength, the kinetics of cytochrome-c2 oxidation induced by a single turnover flash was very different, depending on the concentration of cytochrome c2: at concentrations lower than 1 microM, the process was strictly bimolecular (second-order rate constant, k = 1.7 x 10(9) M-1 s-1), while at higher concentrations a fast oxidation process (half-time lower than 20 microseconds) became increasingly dominant and encompassed the total process at a cytochrome c2 concentration around 10 microM. From the concentration dependence of the amplitude of this fast phase an association constant for a reaction-center--cytochrome-c2 complex of about 10(5) M-1 was evaluated. From the fraction of photo-oxidized reaction centers promptly re-reduced in the presence of saturating concentrations of externally added cytochrome c2, it was found that in approximately 60% of the centers the cytochrome-c2 site was exposed to the external compartment. 3. Both the second-order oxidation reaction and the formation of the reaction-center--cytochrome-c2 complex were very sensitive to ionic strength. In the presence of 180 mM KCl, the value of the second-order rate constant was decreased to 7.0 x 10(7) M-1 s-1 and no fast oxidation of cytochrome c2 could be observed at 10 microM cytochrome c2. 4. The kinetics of exchange of oxidized cytochrome c2 bound to the reaction center with the reduced form of the same carrier, following a single turnover flash, was studied in double-flash experiments, varying the dark time between photoactivations over the range 30 microseconds to 5ms. The experimental results were analyzed according to aminimal kinetic model relating the amounts of oxidized cytochrome c2 and reaction centers observable after the second flash to the dark time between flashes. This model included the rate constants for the electron transfer between the primary and secondary ubiquinone acceptors of the complex (k1) and for the exchange of cytochrome c2 (k2). Fitting to the experimental results indicated a value of k1 equal to 2.4 x 10(3) s-1 and a lower limit for k2 of approximately 2 x 10(4) s-1 (corresponding to a second-order rate constant of approximately 3 x 10(9) M-1 s-1).     

The reaction of cytochrome aa3 with (porphyrin) cytochrome c as studied by pulse radiolysis

(1) Using the pulse-radiolysis and stopped-flow techniques, the reactions of iron-free (porphyrin) cytochrome c and native cytochrome c with cytochrome aa3 were investigated. The porphyrin cytochrome c anion radical (generated by reduction of porphyrin cytochrome c by the hydrated electron) can transfer its electron to cytochrome aa3. The bimolecular rate constant for this reaction is 2 x 10(7) M-1 . s-1 (5 mM potassium phosphate, 0.5% Tween 20, pH 7.0, 20 degrees C). (2) The ionic strength dependence of the cytochrome c-cytochrome aa3 interaction was measured in the ionic strength range between 40 and 120 mM. At ionic strengths below 30 mM, a cytochrome c-cytochrome aa3 complex is formed in which cytochrome c is no longer reducible by the hydrated electron. A method is described by which the contributions of electrostatic forces to the reaction rate can be determined. (3) Using the stopped-flow technique, the effect of the dielectric constant (epsilon) of the reaction medium on the reaction of cytochrome C with cytochrome aa3 was investigated. With increasing epsilon the second-order rate constant decreased.     

Fluorescence polarization and intensity kinetic studies of antifluorescein antibody obtained at different stages of the immune response.

Kinetic studies of reactions between fluorescein and antifluorescein antibody produced during early, intermediate, and late stages of the immune response have been carried out utilizing both fluorescence intensity and polarization measurements in the static (time constant similar to 5 sec) and in the stopped-flow modes (time constant similar to 5 msec). During maturation of the immune response, it was found that the "on" second-order association rate constant increased its value only by a factor of three, whereas the "off" dissociation first-order rate constant decreased by a factor of over 1000. Hence, it is the rate of dissociation which largely determines the stability of the hapten-antihapten complex. Furthermore, since second-order rate behavior was found for even heterogeneous antibody, most of the heterogeneity with respect to binding affinity occurs as a result of the heterogeneity in the rate of dissociation of the hapten-antihapten complex and not from the primary combination of hapten and antibody. Antifluorescein antibody which exhibits both high binding affinity (K similar to 5 x 10(11) M-1) and homogeneity with respect to equilibrium binding has been shown to obey second-order association kinetics over wide ranges in concentration. Despite the fact that the value of the second-order rate constant for this fluorescein-antifluorescein reaction is as large as that for most other hapten-antihapten reactions (1.4 x 10(8) M-1 sec-1), the binding reaction has an appreciable activation energy (7 kcal/mol). This is true for both divalent and univalent antibody. Furthermore, the reaction rate parameters are markedly affected by specific anions. The value of the second-order rate constant (18.5 degrees) increases according to the following scheme: salicylate less than trichloroacetate less than SCN- less than ClO4- less than Cl- less than F- less than phosphate. The activation energy increases as follows: trichloroacetate less than phosphate less than F- less than Cl- less than ClO4- less than SCN- less than salicylate, whereas estimates of the entropy of activation indicate that deltaS++ increases as follows: tricholroacetate less than phosphate similar to F- less than Cl- less than ClO4- less than SCN- less than salicylate. The same mechanism which was previously proposed by us for the antigen-antibody reaction is also consistent with the kinetics of the fluorescein-antifluorescein reaction. This mechanism postulates a bimolecular process with structural rearrangements (conformational changes and/or the loss of water) in the formation of the transition state complex. The reaction between the fluorescein hapten and its antibody hence is not diffusion limited.