One of the longest and most intensively studied enzymes is horseradish peroxidase. Its reactions are reviewed from historical, kinetic and mechanistic perspectives. Kinetics studies include steady state, transient state and relaxation kinetics. The methods and reasoning involved are applicable to other peroxidases and indeed to other enzymes. Accumulated evidence from several techniques indicate that distal His 42 plays a key role in its redox reactions. The possible implications of recent low temperature neutron diffraction experiments on oxidized yeast cytochrome c peroxidase are discussed.

Two specific mechanisms are proposed to explain the effect of buffer on the reactions catalysed by fumarase. In one mechanism, buffer catalyses a slow isomerisation of the enzyme in which a catalytic acid group on one side of the active site is replaced by another acid group on the opposite side. In the other mechanism the buffer-catalysed isomerisation reaction is fast. The latter mechanism fits the experimental data.

The pH range for Compound I formation of horseradish peroxidase (2.5 to 11) is the largest for any known enzyme reaction. A key part of the reaction is proton transfer from hydrogen peroxide to distal His42. This proton is retained to complete formation of a water leaving group as the ferryl porphyrin π-cation radical is formed. How can the imidazolium side chain of His42 retain a proton at very high pH? And how can it give up the proton when required at very low pH? The answer is rearrangement of electronic charge through Electron Density Circuits (EDCs) in the protein matrix. An increase of at least 9 p K a units, which occurs on the imidazole side chain of His42 as the Compound I reactive intermediate is formed, is facilitated by an EDC. A reverse EDC facilitates proton transfer to form the water leaving group. The pathway of the EDC involves the heme, its propionate side chains, Arg41, and His42. The occurrence of EDCs in chymotrypsin reactions reinforces the nucleophilic attack, and the subsequent electron and proton transfers. They also can shift p K a values. The catalytic triad of chymotrypsin is Ser195, His57, and Asp102. The currently accepted one-proton transfer mechanism involves Ser195 and His57, with Asp102 being regarded as too acidic to accept a proton. A comparatively small shift in the p K a value of Asp102 is all that is required to make it a proton acceptor from His57 so a two-proton mechanism may occur.

PREFACE. CONTRIBUTORS. 1 HISTORICAL: PIONEERING WORK ON HORSERADISH AND YEASTCYTOCHROME c PEROXIDASES. Introduction. Techniques and Instrumentation. Summary and Conclusions. References. 2 HEME PEROXIDASE AND CATALASE FAMILIES. Plant, Fungal, and Bacterial Peroxidases. Mammalian Peroxidases. Catalases. References. 3 HORSERADISH PEROXIDASE. I. THE NATIVE ENZYME, COMPOUNDS IAND II, THEIR STRUCTURES, AND THEIR CYCLE. Introduction. The Classic Peroxidase Cycle. Structure and Properties of Native Horseradish Peroxidase C. Horseradish Peroxidase Compound I (HRP-I). Horseradish Peroxidase Compound II (HRP-II). Some Diverse Approaches to an Understanding of HorseradishPeroxidase. References. 4 HORSERADISH PEROXIDASE. II. TWO-ELECTRON REACTIONS, FERROUSPEROXIDASE, COMPOUND III, THE FIVE OXIDATION STATES, OXYGENEVOLUTION, AND INACTIVATION. Introduction. Two-Electron Oxidations By Compound I. Oxygen Transfer By One-Electron Mechanisms. Ferrous Horseradish Peroxidase and Compound III. The Five Oxidation States of Horseradish Peroxidase. The Catalatic Reaction. The HRP Clock Reaction. Enzyme Inactivation. References. 5 HORSERADISH PEROXIDASE. III. OSCILLATIONS ANDPEROXIDASE-OXIDASE REACTIONS WITH NADH, INDOLE-3-ACETIC ACID, ANDISOBUTYRALDEHYDE. LIGHT EMISSION. Oscillations and the NADH Peroxidase-Oxidase Reaction. Peroxidase Oxidase Reaction with Indole-3-Acetic Acid. Reaction of Isobutyraldehyde with Horseradish Peroxidase. References. 6 YEAST CYTOCHROME c PEROXIDASE: REACTIONS WITH SMALLSUBSTRATES. Introduction. Properties of Yeast Cytochrome c Peroxidase. Crystal Structures of Yeast Cytochrome c Peroxidase, itsCompounds and Complexes. Mechanism of Compound I Formation. The Reaction Cycle for Yeast Cytochrome c Peroxidase. Steady-State Kinetics. References . 7 YEAST CYTOCHROME c PEROXIDASE: REACTION WITHCYTOCHROME c. Introduction. Experimental Results. References. 8 SPECTROSCOPY. I. OPTICAL, RESONANCE RAMAN, AND X-RAYABSORPTION. Optical Absorption Spectra. Resonance Raman Spectra. X-ray Absorption Spectroscopy. References. 9 SPECTROSCOPY. II. NUCLEAR MAGNETIC RESONANCE, ELECTRONSPIN, AND MOSSBAUER. Nuclear Magnetic Resonance (NMR) Spectroscopy. Electron Spin Resonance (ESR) Spectroscopy. Mossbauer Spectroscopy. References. 10 THEORETICAL. Peroxidase Kinetics. Marcus Theory for Electron Transfer Reactions. Electron Tunneling. Electron Transfer Reactions in Proteins. Electron Density Circuits. Diffusion Control. Quantum Mechanical Calculations. References. 11 CLASS I: ASCORBATE PEROXIDASE. Introduction. Sequencing and Cloning. Properties, Reactions, and Intermediate Compounds. Crystal Structures. References. 12 CATALASE-PEROXIDASES AND MYCOBACTERIUMTUBERCULOSIS. Introduction. Structures of Catalase-Peroxidases. Isoniazid and Other Reactants of Catalase-Peroxidases. The Oxidative Defense Mechanisms of MycobacteriumTuberculosis. References. 13 CLASS II. LIGNIN, MANGANESE, VERSATILE, AND COPRINUSCINEREUS PEROXIDASES. Lignin Peroxidase. Manganese Peroxidase. Other Manganese Peroxidases, Versatile Peroxidase. Coprinus Cinereus (Arthromyces Ramosus) Peroxidase. References. 14 OTHER CLASS III PEROXIDASES. Arabidopsis Thaliana Peroxidase. Barley Peroxidase. Peanut Peroxidase. Soybean Peroxidase. Tobacco Peroxidases. Turnip Peroxidases. References. 15 CATALASES (Peter Jones). Introduction. Perspective. Progress. Catalases in Biology. Prospects. References. 16 MYELOPEROXIDASE: ENZYMOLOGY. Introduction. Properties of Myeloperoxidase. The Compounds of Myeloperoxidase. Reactions of Myeloperoxidase. Cloning of Myeloperoxidase: Site-Directed Mutagenesis. The Crystal Structure and the Prosthetic Group ofMyeloperoxidase. Eosinophil Peroxidase. References. 17 BIOMEDICAL ASPECTS OF MYELOPEROXIDASE: HALOGENATIONREACTIONS IN CARDIOVASCULAR DISEASE, INFECTION, AND CANCER(Jeffrey P. Henderson and Jay. W. Heinecke). Introduction. Oxidants Produced by MPO in Humans. MPO and Coronary Artery Disease. MPO and Carcinogenesis. Prospects. References. 18 PROSTAGLANDIN H SYNTHASE. Introduction. Crystal Structures. Prostaglandin H Synthase-2. Preliminary Mechanistic Studies. Detection of Free Radicals: Role of ESR Spectroscopy. The Role of Aspirin and Related Substances: Contributions ofVane and Smith. Work of Marnett and Coworkers. Work of Kulmacz, Tsai, and Coworkers. Manganese Prostaglandin Synthases. Mechanistic Details. References. 19 THYROID PEROXIDASE. Introduction. Hormone Discovery and Chemical Synthesis. Detection of the Method of Biological Synthesis ofThyroxine. Conclusions. References. 20 LACTO- AND SALIVARY PEROXIDASES. Introduction. Properties. The Compounds of Lactoperoxidase and Their Reactions. References. 21 CHLOROPEROXIDASE FROM C. FUMAGO. Introduction. History. Optical Spectra. ESR, Endor, Mossbauer, Exafs, and Resonance RamanSpectra. Investigations of Compounds I and II. Structure of Compound I and the Catalatic Reaction. Ligand Binding. Kinetics and Mechanisms of Chlorination and Oxidation. Amino Acid Sequence and Crystal Structure. References. 22 SELENIUM-CONTAINING ENZYMES: GLUTATHIONE PEROXIDASE ANDIODOTHYRONINE DEIODINASE. Introduction. Glutathione Peroxidase. Iodothyronine Deiodinase. References. 23 STRUCTURE AND FUNCTION OF VANADIUM HALOPEROXIDASES(Ron Wever and Rokus Renirie). Summary. Abbreviations. Introduction. Occurrence and Biological Function of Vanadium Iodo- andBromoperoxidases. Occurrence and Biological Function of VanadiumChloroperoxidases. Catalytic Properties of Bromoperoxidase. Properties of the Prosthetic Group in Bromoperoxidase. Kinetic and Optical Properties of VanadiumChloroperoxidases. Sulfoxidation Reactions. Stability of Bromo- and Chloroperoxidases. X-ray Structures of Vanadium Bromoperoxidases. Active Site of Vanadium Bromoperoxidase From A.Nodosum. X-ray Structures of the Vanadium Chloroperoxidase and Details ofthe Active Site. X-ray Structure of the Peroxo-Intermediate of VanadiumChloroperoxidase and Difference in Reactivity Between Chloro- andBromoperoxidases. Nature of the Vanadate Cofactor. References. 24 OTHER HEME PEROXIDASES AND ENZYMES. DI-Heme Peroxidases. Peroxidases Everywhere You Look. Myoglobins. Hemoglobin. Cytochrome c Oxidase. Oxygenases. Heme Oxygenase. Guanylyl Cyclase. References. 25 APPLICATION OF PEROXIDASES (Ron Wever). Introduction. Background Information. Peroxidases as Pharmaceutical and/or Antimicrobial Agents. Applications in Bleaching and Detergents. Biotransformations. Polymerization Reactions and Wastewater Purification. Depolymerization Reactions. Analytical Applications. Medical Applications. References. AUTHOR INDEX. SUBJECT INDEX.

The most recently proposed mechanisms for the formation of the Compound I intermediates of the peroxidases and catalases have been based on the crystallographic elucidation of the enzyme structures. It has been assumed that these mechanisms are compatible with an earlier proposal of the formation of a reversible enzyme-substrate intermediate called Compound 0, which was based on data that pre-dated the availability of the enzyme structures. However, it is argued here that this is not the case and some modifications of the existing mechanism are proposed which reconcile the structural, kinetic and energetic data for the reactions. This paper focuses attention on horseradish peroxidase isoenzyme C and particularly on the acid-base properties of the imidazole side chain of distal histidine 42. This imidazole group has an exceptionally low pK(a) value in the resting enzyme, which is higher in Compound I and higher still in Compound II. The pK(a) value must also be greatly increased following Compound 0 formation so that the imidazole can become an effective proton acceptor. An explanation is offered in a dielectric insertion (DI) model, in which the peroxide substrate, or fragments thereof, screens the influence of the positively charged heme iron on the pK(a) value of the imidazole group. It is proposed that Compound 0 is converted to a second intermediate, Compound 0*, by intramolecular proton transfer along a pre-existing hydrogen bond, a process which reduces the energy requirements of charge separation in the deprotonation of hydrogen peroxide.

The kinetics and mechanism of action of the most intensively studied mammalian peroxidases, myeloperoxidase and prostaglandin H synthase are critically reviewed. Evidence against currently favored mechanisms is presented. It is shown that myeloperoxidase has a strong defence mechanism against free hypochlorous acid, commonly thought to be its principal product in its bactericidal activity. Rather, after its two-electron oxidation of chloride ion, myeloperoxidase rapidly converts it into an enzyme-bound chlorinating intermediate, most likely a chlorinated distal imidazole ring. This species chlorinates taurine which may either be a transfer agent of Cl+ to other species or may act directly in attack on invading microorganisms. The currently favored mechanism of action of prostaglandin H synthase-1 is a branching chain mechanism in which Compound I is converted into a species containing a tyrosyl radical on the opposite side of the enzyme. Once the tyrosyl radical is formed it converts arachidonic acid into a peroxide in a cyclooxygenase reaction, independent of the peroxidase activity. This mechanism cannot explain the enhancing effect of small free radical scavengers, nor the fact that peroxidase activity continues unabated while the cyclooxygenase reaction is proceeding, nor the 2: 1 ratio of small free radical scavenger to arachidonic acid consumption. A tightly coupling of peroxidase and cycloxygenase reactions appears to be the steady state mechanism, and the branching chain mechanism, if it occurs, is confined to a burst transient state phase.

aerobic oxidation of IAA produces an electronical1y excited The inhibition of horseradish peroxidase (HRP)-cataspeci~s. The c~~miluminescence of these spe~ies and th~ir lyzed oxidation of inciole-3-acetic acid (IAA) by a phenol, chemIcal reactivity have been thoroughly studied by G. CIIcaffeic acid (CA)~ w~ stndied nsing both a kinetic apento a~d co-wor~er~/or ma~y yea~s. The~ sugg~sted,~ ~yproach and compnt~r simulation. The presence of CA pothesl~ to ex~l~n photoblOc~em..stry without lIght,. dlsresnlted in a lag period in IAA oxidation. The lag period c~ssed In det~l In a number of reV1ews (2-5). AccordIng to increased slowly with increasing [CA] nntil a critical con~hlS hypothesIs the energy of an enzyme-generated electroncentration [CA] was reached then it increased much lcally excited species can give rise to photochemical-like faster wh;n [CAj' was greater'than [CA] The [CA] reactions in living systems in the dark. The intriguing results was proportional to' [lAA] and did not depend upo: of Cilento's group stimulated a study of different aspects of [HRP]. Caffeic acid was oxidized by compound I and IAA oxidation in our groups (in Russia and in Canada). One compound II of HRP with bimolecular rate constants (6.8 of us (H.B.D.) collaborated with G. Cilento for many years. :!: 107 and 2.1 :!: 107 M-IS-I)~ which were much higher Although progress in understanding the mechanisms of perthan the corresponding rate constants for IAA oxidation oxidase-catalyzed lAA oxidation and related processes have (2.3 :!: 103 and 2.0 :!:; 102 M-1S-1). Our experimental data been achieved, many questions remain unresolved. show that CA inhibits IAA oxidation because it is able The IAA/horseradish peroxid8se (HRP)/Oz ~Y!ltem emits to compete effectively as a peroxidase substrate. A model in three spectral regions: 420, 465 and 535 nm with different based on a detailed mechanism of IAA oxidation was inbehavior exhibited in each region (6-8). Therefore at least vestigated using computer simulation. A rate constant three different electronically excited products (intermediates) driving nonenzymatic hydroperoxide formation in IAA are fonned (6). Identification of these products is a complex solution was determined, 3.0 x 10-7 S-1. The model quanbut solvable problem. The yields of these products are very titatively describes ~e experimental results of this work sensitive to experimental conditions, in particular, pH. At and also qualitatively explains data published earlier. neutral pH, and under steady-state conditions of IAA oxiThe critical inhibitor concentration is approximately dation, only the product emitting at 420 nm is formed (6). equal to twice the concentration of hydroperoxide in IAA De Mello et al. (8) suggested that hydroperoxide, ROOH, solution at the time iof inhibitor addition. Therefore hywhich is an intermediate of IAA oxidation, is responsible for droperoxide concentration can be calculated from the dethis emission. There is indirect evidence for this hypothesis. termination of critical inhibitor concentration. Oxidation of IAA at neutral pH goes through a peroxidase pathway accompanied by a free radical chain reaction (9,10). INTRODUCTION' Our computer simulation showed that ROOH is the intermediate of highest concentration, and it reaches its steady'/". Indole-3-acetic acid (IAA):(: is a natural phytohormone with state concentration much later than the other intermediates : many growth regulatory functions. The level of IAA in (10 min and I min, respectively) (11). Preliminary results Ii plants is, in particular, controlled via its enzymatic oxidation show that the intensity of the 420 nm ~hemi1uminescence J~ that is catalyzed by peroxidases (1). Peroxidase-catalyzed from the IAAlHRP/O2 system also reaches the steady state after a long lag period (6,12). However, in order to prove a *This paper is dedicatecllo the memory of Giuseppe Cilento. direct connection between ROOH concentration and che,To whom CoJTespoodence should be addressed at: Department of miluminescence at 420 nm, the time dependence of ROOH Chemistry, University of Alberta, Edmonton, Alberta T6G 2G2, has to be measured experimentally. A simple indirect way Canada. Fax: 403-492-8231: e-mail: brian.dunford@ualberta.ca f ROOH . h I +A bb . t CA ff . .d HRP HRP I d HRP II . 0 measurement In t e AA/HRP/O2 system was sug1-" revla Ions: , ea elc act; , ao ,native .. . Conn, compound I and compound II of horseradish peroxidase, gested by Nakajima and Yamazaki (9). They used ascorbate respectively; IAA and RH, indole-3-acetic acid. to induce a lag period in lAA oxidation. They proposed that @ 1996 American Society for Photobiology 0031-8655196 $5.00+0.00 the critical concentration of ascorbate causing a sudden in735-741

The mechanism of reaction of hexaquo iron(II) with hydrogen peroxide has been unresolved for 70 years. Most scientists, perhaps by default, have accepted the free radical chain mechanism of Barb et al. (Trans. Faraday Soc. 47 (1957) 462). However an earlier proposal involved formation of the ferryl ion, FeO2+ (J. Am. Chem. Soc. 54 (1932) 2124). Recent work has favored a mechanism involving FeO2+ and FeOFe5+ species. Similarly there are differences of opinion on the mechanism of reaction of iron(III), both hexaquo and chelated, with hydrogen peroxide. These differences have fostered a recent burst of activity, with claims on one hand that hydroxyl radicals play a key role, and on the other, that there is a non-free radical mechanism. In contrast, the mechanism of reaction of the heme-containing peroxidase and catalase enzymes with hydrogen peroxide, orders of magnitude faster than reactions of iron(II)/(III), now appears to be well established. In this paper I attempt, as objectively as possible, to delineate the proposed mechanisms, discuss their possible physiological relevance, and summarize the current state of knowledge.

AbstractThe oxiding properties of metalloenzymes are reviewed. The reactive oxygen‐containing species formed by successive one‐electron reductions of dioxygen to water are discussed. Emphasis is on iron‐containing enzymes and in particular heme enzymes. Details of peroxidase, catalase, cytochrome P450, nitric oxide synthase, guanylate cyclase, and cytochrome c oxidase reactions are summarized. A brief sojourn looks at the role of sulfur, which converts iron‐containing enzymes from oxidation catalysts to reduction catalysts. An example is nitrogenase where iron–sulfur clusters contribute to the molybdenum‐catalyzed reduction of dinitrogen to ammonia. Copper enzymes and in particular copper‐zinc superoxide dismutase are also examined.

Binding of p-cresol to native horseradish peroxidase was investigated by differential spectrophotometry, and the value lo3 Kdiss = 3 M was obtained at neutral and acid pH; binding is not competitive with that of cyanide and hydroxide. The Soret region spectrum of Compound II of the enzyme was measured in the steady state at pH 4.26, 6.89, and 10.95, and the differences were found to be too small to be attributed to the acid dissociations of an iron-bound group. The kinetics of the reactions between Compound II and p-cresol, ferrocyanide, and iodide was investigated in 94% DzO. Almost no solvent isotope effect was found on the ionization of the group of pKa 8.6. The reaction with p-cresol gave kD/kX = 0.45 f 0.04, which was attributed to a rate-determining proton transfer. In regions of the pH log rate profile having a slope of -1 ferrocyanide gave kDlkH = 2.6 f 0.3 at high pH and 2.9 rt 0.6 at low pH, whereas iodide gave kD/kH = 4.0 f 0.4. The pH rate profiles, isotope effects, and over-all rates of the three reactions were correlated in terms of a mechanism involving intramolecular general acid catalysis. In sufficiently acidic solutions this ultimately gives way to a specific acid-catalyzed mechanism, and the mechanistic changeover occurs at a higher pH the more difficult the over-all reaction.

. The effect of electron flow through a complete circuit on transition state acid dissociation constants is used to explain the remarkable catalysis observed in a redox reaction, the formation of compound I from native peroxidase. The explanation for the huge shift in the dissociation constant of a distal histidine residue, in going from the resting enzyme to the transition state, is a complete electron circuit through many amino acid residues and hydrogen bonds which prevents the development of localized charge. The key feature is electron flow through the circuit at the instant that proton transfer is occurring in the opposite direction. Electron flow occurs in one direction for attainment of the transition state and in the opposite direction for product formation.

The first complete mechanistic analysis of halide ion oxidation by a peroxidase was that of iodide oxidation by horseradish peroxidase. It was shown conclusively that a two-electron oxidation of iodide by compound I was occurring. This implied that oxygen atom transfer was occurring from compound I to iodide, forming hypoiodous acid, HOI. Searches were conducted for other two-electron oxidations. It was found that sulfite was oxidized by a two-electron mechanism. Nitrite and sulfoxides were not. If a competing substrate reduces some compound I to compound II by the usual one-electron route, then compound II will compete for available halide. Thus compound II oxidizes iodide to an iodine atom, I-., although at a slower rate than oxidation of I- by compound I. An early hint that mammalian peroxidases were designed for halide ion oxidation was obtained in the reaction of lactoperoxidase compound II with iodide. The reaction was accelerated by excess iodide, indicating a co-operative effect. Among the heme peroxidases, only chloroperoxidase (for example from Caldariomyces fumago) and mammalian myeloperoxidase are able to oxidize chloride ion. There is not yet a consensus as to whether the chlorinating agent produced in a peroxidase-catalyzed reaction is hypochlorous acid (HOCl), enzyme-bound hypochlorous acid (either Fe-HOCl or X-HOCl where X is an amino acid residue), or molecular chlorine Cl-2. A study of the nonenzymatic iodination of tyrosine showed that the iodinating reagent was either HOI or I,. It was impossible to tell which species because of the equilibria:I-2 + H2O = HOI + I- + H+I= + I-2 = I-3(-)The same considerations apply to product analysis of an enzyme-catalyzed reaction. Detection of molecular chlorine Cl-2 does not prove it is the chlorinating species. If Cl-2 is in equilibrium with HOCl then one cannot tell which (if either) is the chlorinating reagent. Examples will be shown of evidence that peroxidase-bound hypochlorous acid is the chlorinating agent. Also a recent clarification of the mechanism of reaction of myeloperoxidase with hydrogen peroxide and chloride along with accurate determination of the elementary rate constants will be discussed.

The kinetics and spectra of the reactions of clozapine with compounds I and II of myeloperoxidase were investigated using both single- and sequential-mixing stopped-flow techniques, steady-state kinetics, and spectrophotometric measurements. The results show conclusively that both compounds I and II are reduced in one-electron reactions with clozapine. At pH 7.0 the rate constant for compound I reacting with clozapine is (1.5 +/- 0.1) x 10(6) M(-1) s(-1) and for compound II (4.8 +/- 0.1) x 10(4) M(-1) s(-1). The physiological pH of 7.4 was found to be optimal for the oxidation of clozapine by compound I. The rate constant for compound I reacting with ascorbate is (1.1 +/- 0.1) x 10(6) M(-1) s(-1) and for compound II (1.1 +/- 0.2) x 10(4) M(-1) s(-1), both obtained at pH 7.0. Experiments with both clozapine and ascorbate present showed that ascorbate acts both as a competitive inhibitor and free radical scavenger.

The oxidation of serotonin (5-hydroxytryptamine) by the myeloperoxidase intermediates compounds I and II was investigated by using transient-state spectral and kinetic measurements at 25.0 +/- 0.1 degrees C. Rapid scan spectra demonstrated that both compound I and compound II oxidize serotonin via one-electron processes. Rate constants for these reactions were determined using both sequential-mixing and single-mixing stopped-flow techniques. The second order rate constant obtained for the one-electron reduction of compound I to compound II by serotonin is (1.7 +/- 0.1) x 10(7) M(-1) x s(-1), and that for compound II reduction to native enzyme is (1.4 +/- 0.1) x 10(6) M(-1) x s(-1) at pH 7.0. The maximum pH of the compound I reaction with serotonin occurs in the pH range 7.0-7.5. At neutral pH, the rate constant for myeloperoxidase compound I reacting with serotonin is an order of magnitude larger than for its reaction with chloride, (2.2 +/- 0.2) x 10(6) M(-1) x s(-1). A direct competition of serotonin with chloride for myeloperoxidase compound I oxidation was observed. Our results suggest that serotonin may have a role to protect lipoproteins from oxidation and to prevent enzymes from inactivation caused by the potent oxidants HOCl and active oxygen species.

Interrelations between peroxidase and cyclooxygenase reactions catalyzed by prostaglandin endoperoxide synthase (prostaglandin H synthase) were analyzed in terms of the mutual influence of these reactions. The original branched-chain mechanism predicts competition between these two reactions for enzyme, so that peroxidase cosubstrate should inhibit the cyclooxygenase reaction and the cyclooxygenase substrate is expected to inhibit the peroxidase reaction. In stark contrast, the peroxidase reducing substrate is well known to strongly stimulate the cyclooxygenase reaction. In the present work the opposite effect, the influence of the cyclooxygenase substrate on the peroxidase reaction was studied. Experiments were conducted on the effect of arachidonic acid on the consumption of p-coumaric acid by prostaglandin H synthase and 5-phenyl-4-pentenyl-1-hydroperoxide. Neither the steady-state rates nor the total extent of p-coumaric acid consumption was affected by the addition of arachidonic acid. This suggests that the cyclooxygenase substrate does not influence observable velocities of the peroxidase reaction, namely oxidation and regeneration of the resting enzyme. The data support coupling of the cyclooxygenase and peroxidase reactions. A combination of the branched-chain and tightly coupled mechanisms is proposed, which includes a tyrosyl radical active enzyme intermediate regenerated through the peroxidase cycle. Numerical integration of the proposed reaction scheme agrees with the observed relations between peroxidase and cyclooxygenase reactions in the steady state.

The peroxidase reaction of prostaglandin endoperoxide synthase was investigated by transient state kinetics using stoichiometric amounts of substrates. The rate constants for the conversion of compound I to intermediate II determined with a stoichiometric amount of hydroperoxide were found to be lower by an order of magnitude than when an excess of hydroperoxide was used. The difference was attributed to ability of the compound I of prostaglandin endoperoxide synthase to be reduced by the excess of hydroperoxide. This suggests that the true rate constant of unimolecular conversion compound I to intermediate II at 3 degrees C is 5-10 s-1 instead of 50-200 s-1 as reported before. The latter value rather characterizes the combined process of spontaneous and hydroperoxide-dependent transformation of compound I. Stoichiometric amounts of reducing substrates significantly stimulated transformation of compound I. This effect could not be entirely explained by their reducing action, which was measured by following the oxidation kinetics. The results of the global fit of the experimental data suggest that reducing substrates, in addition to their direct action in reducing compound I to compound II, indirectly stimulate transformation of compound I to the tyrosyl radical form of intermediate II, thereby stimulating the cyclooxygenase reaction.

In this study the reactions between nitric oxide (NO) and horseradish peroxidase (HRP) compounds I and II were investigated. The reaction between compound I and NO has biphasic kinetics with a clearly dominant initial fast phase and an apparent second-order rate constant of (7.0 +/- 0.3) x 10(5) M(-1) s(-1) for the fast phase. The reaction of compound II and NO was found to have an apparent second-order rate constant of k(app) = (1.3 +/- 0.1) x 10(6) M(-1) s(-1) or (7.4 +/- 0.7) x 10(5) M(-1) s(-1) when measured at 409 nm (the isosbestic point between HRP and HRP-NO) and 419 nm (lambda(max) of compound II and HRP-NO), respectively. Interestingly, the reaction of compound II with NO is unusually high relative to that of compound I, which is usually the much faster reaction. Since horseradish peroxidase is prototypical of mammalian peroxidases with respect to the oxidation of small substrates, these results may have important implications regarding the lifetime and biochemistry of NO in vivo after inflammation where both NO and H(2)O(2) generation are increased several fold.

The base analog, 2-aminopurine (2AP), was used as a fluorescent reporter of the biochemical steps in the proofreading pathway catalyzed by bacteriophage T4 DNA polymerase. "Mutator" DNA polymerases that are defective in different steps in the exonucleolytic proofreading pathway were studied so that transient changes in fluorescence intensity could be equated with specific reaction steps. The G255S- and D131N-DNA polymerases can hydrolyze DNA, the final step in the proofreading pathway, but the mutator phenotype indicates a defect in one or more steps that prepare the primer-terminus for the cleavage reaction. The hydrolysis-defective D112A/E114A-DNA polymerase was also examined. Fluorescent enzyme-DNA complexes were preformed in the absence of Mg2+, and then rapid mixing, stopped-flow techniques were used to determine the fate of the fluorescent complexes upon the addition of Mg2+. Comparisons of fluorescence intensity changes between the wild type and mutant DNA polymerases were used to model the exonucleolytic proofreading pathway. These studies are consistent with a proofreading pathway in which the protein loop structure that contains residue Gly255 functions in strand separation and transfer of the primer strand from the polymerase active center to form a preexonuclease complex. Residue Asp131 acts at a later step in formation of the preexonuclease complex.