Cyanide binding to prostaglandin H (PGH) synthase results in a spectral shift in the Soret region. This shift was exploited to determine equilibrium and kinetic parameters of the cyanide binding process. At pH 8.0, ionic strength 0.22 M, 4 degrees C, the cyanide dissociation constant, determined from equilibrium experiments, is (65 +/- 10) microM. The binding rate constant is (2.8 +/- 0.2) x 10(3) M-1 s-1, and the dissociation rate constant is zero within experimental error. Through a kinetic study of the binding process as a function of pH, from pH 3.96 to 8.00, it was possible to determine the pKa of a heme-linked acid group on the enzyme of 4.15 +/- 0.10 with citrate buffer. An apparent pKa of 4.75 +/- 0.03 was determined with acetate buffer; this different value is attributed to complexation of the enzyme with one of the components of the acetate buffer.

AbstractThe fused cyclobutanol acetate (I) is hydrolyzed and directly coupled with the isocyanate (II), yielding the urethane (III).

The spectral behavior of the enzyme prostaglandin H synthase was studied in the Soret region under conditions that permitted comparison of enzyme intermediates involved in peroxidase and cyclooxygenase activities. First, the peroxidase activity was examined. The enzyme's spectral behavior upon reacting with 5-phenyl-pent-4-enyl-1-hydroperoxide was different depending on the presence or absence of the reducing substrate, phenol. In the reaction of prostaglandin H synthase with the peroxide in the absence of phenol, formation of the enzyme intermediate compound I is observed followed by partial conversion to compound II and then by enzyme bleaching. In the reaction with both peroxide and phenol the absorbance decreases and a steady-state spectrum is observed which is a mixture of native enzyme and compound II. The steady state is followed by an increase in absorbance back to that of the native enzyme with no bleaching. The difference can be explained by the reactivity of phenol as a reducing substrate with the prostaglandin H synthase intermediate compounds. Cyclooxygenase activity with arachidonic acid could not be examined in the absence of diethyldithiocarbamate because extensive bleaching occurred. In the presence of diethyldithiocarbamate, enzyme spectral behavior similar to that seen in the reaction of the peroxide and phenol was observed. The similarity of the spectra strongly suggests that the enzyme intermediates involved in both the peroxidase and cyclooxygenase reactions are the same.

A combination of cyclooxygenase activity assays, rapid spectrophotometry and presteady-state, steady-state, and transient-state kinetics is used to characterize further the properties of prostaglandin H synthase. 13-Hydroperoxyoctadeca-9-11-dienoic acid is used as oxidizing substrate and the effects of the following compounds are examined: arachidonic acid, N,N,N′,N′-tetramethyl-p-phenylenediamine, phenol, diethyldithiocarbamate, and the nonsteroidal anti-inflammatory drugs aspirin, indomethacin, phenylbutazone, and Bromfenac. The order of reactivity of four of these substrates, predominantly with compound II of prostaglandin H synthase, is N,N,N′,N′-tetramethyl-p-phenylenediamine > phenol > indomethacin ∼ phenylbutazone. Aspirin exhibits no effect. Arachidonic acid causes inactivation. Diethyldithiocarbamate acts as a reducing substrate for the oxidized forms of prostaglandin H synthase. Bromfenac appears to act both as a protective agent and inhibitor.

The mechanism of malonaldehyde oxidation by horseradish peroxidase in the presence of manganese(II) and acetate was investigated. Our results show that an apparent oxygenase behavior demonstrated by peroxidase in this system can be explained in terms of normal peroxidase activity. A free radical is generated from the reaction of malonaldehyde with compounds I and II of peroxidase; this radical is scavenged by dissolved molecular oxygen to give the appearance of peroxidase acting as an oxygenase. Oxygen consumption, absorbance spectra, and kinetic results show that the reaction is initiated by autoxidation of malonaldehyde to give a free radical. The radical reacts with oxygen and through the action of manganese(II), a peroxide is generated. This peroxide drives the peroxidase cycle to generate more free radicals which propagate the oxygen consumption reaction.

Elementary reactions have been studied quantitatively in the complex overall process catalyzed by horseradish peroxidase whereby isobutyraldehyde and molecular oxygen react to form triplet state acetone and formic acid. The rate constant for the reaction of the enol form of isobutyraldehyde with compound I of peroxidase is (8 +/- 1) X 10(6) M-1 s-1 and with compound II (1.3 +/- 0.3) X 10(6) M-1 s-1. Neither the enolate anion nor the keto form is reactive. The reactivity of enols with peroxidase parallels that of unionized phenols and a common mechanism is proposed. The overall catalyzed reaction of isobutyraldehyde and oxygen consists of an initial burst followed by a steady state phase. The burst is caused by the following sequence: 1) an initial high yield of compound I is formed from reaction of native enzyme with the autoxidation product of isobutyraldehyde, a peracid and 2) compound I rapidly depletes the equilibrium pool of enol which is present. After this burst a steady state phase is observed in which the rate-limiting step is the conversion of the keto to the enol form of the aldehyde catalyzed by phosphate buffer. The rate constant for the keto form reacting with phosphate is (8.7 +/- 0.6) X 10(-5) M-1 s-1. All constants were measured in dilute aqueous ethanol at 35 degrees C, pH 7.4, and ionic strength 0.67 M. Both the initial burst of light and the steady state emission from triplet acetone can be observed with the naked eye. Since the magnitude of the burst is a measure of the equilibrium amount of enol, the keto-enol equilibrium constant is readily calculated and hence also the rate constant for conversion of enol to keto. The keto-enol equilibrium constant is unaffected by phosphate which therefore acts as a true catalyst.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTMeasurement of rates and equilibria for keto-enol tautomerism of aldehydes using horseradish peroxidase compound ICornelia. Bohne, I. David. MacDonald, and H. Brian. DunfordCite this: J. Am. Chem. Soc. 1986, 108, 24, 7867–7868Publication Date (Print):November 1, 1986Publication History Published online1 May 2002Published inissue 1 November 1986https://doi.org/10.1021/ja00284a080RIGHTS & PERMISSIONSArticle Views229Altmetric-Citations15LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (285 KB) Get e-Alertsclose Get e-Alerts