Douglas C. Goodwin

Arachidonic Acid Oxygenation by COX-1 and COX-2 MECHANISMS OF CATALYSIS AND INHIBITION

Prostaglandins were discovered in human semen in 1930, but their low concentrations and instability precluded identification for nearly 30 years (for a brief historical review, see Ref. 1). Once they were identified, it was clear they arose from polyunsaturated fatty acids by a complex series of reactions involving oxygenation, cyclization, and the generation of five chiral centers from an achiral substrate. The mechanism of prostaglandin biosynthesis was outlined in 1967 by Hamberg and Samuelsson (2), and the basic tenets have been confirmed in subsequent studies. The key step in their proposed mechanism was the formation of bicyclic peroxides (endoperoxides) as the initial products of polyunsaturated fatty acid oxygenation (Fig. 1). The term cyclooxygenase (COX) 2 was coined to describe the enzyme that carried out this complex chemical transformation, and its role was confirmed by the isolation of prostaglandin endoperoxides in 1973 (3, 4). In addition to catalyzing a fascinating metabolic transformation, COX is an enormously important pharmacological target. Vane reported in 1971 (5) that non-steroidal anti-inflammatory drugs (NSAIDs) inhibit prostaglandin formation and demonstrated that their relative inhibitory potency in vitro correlates to their antiinflammatory activity in vivo. This not only explained the beneficial activity of NSAIDs but also their side effects such as gastrointestinal toxicity and bleeding because prostaglandins and related molecules (i.e. thromboxane) are involved in a very broad range of physiological and pathophysiological responses. The importance of these molecules as autocrine and paracrine mediators has been confirmed recently by the phenotypes of mice bearing targeted deletions in COX genes or prostaglandin receptor genes. The discovery of a second gene (COX-2) coding for cyclooxygenase and the demonstration that its protein product is distributed differently from the originally discovered enzyme (COX-1) raised the possibility that some of the beneficial effects of NSAIDs may be separable from their side effects by development of isoform-selective inhibitors (6–9). This hypothesis has been dramatically validated by the demonstration that selective COX-2 inhibitors are anti-inflammatory and analgesic but lack the gastric toxicity associated with all currently available NSAIDs (10, 11).

Effects of nitric oxide and nitric oxide-derived species on prostaglandin endoperoxide synthase and prostaglandin biosynthesis

Prostaglandins and NO· are important mediators of inflammation and other physiological and pathophysiological processes. Continuous production of these molecules in chronic inflammatory conditions has been linked to development of autoimmune disorders, coronary artery disease, and cancer. There is mounting evidence for a biological relationship between prostanoid biosynthesis and NO· biosynthesis. Upon stimulation, many cells express high levels of nitric oxide synthase (NOS) and prostaglandin endoperoxide synthase (PGHS). There are reports of stimulation of prostaglandin biosynthesis in these cells by direct interaction between NO· and PGHS, but this is not universally observed. Clarification of the role of NO· in PGHS catalysis has been attempted by examining NO· interactions with purified PGHS, including binding to its heme prosthetic group, cysteines, and tyrosyl radicals. However, a clear picture of the mechanism of PGHS stimulation by NO· has not yet emerged. Available studies suggest that NO· may only be a precursor to the molecule that interacts with PGHS. Peroxynitrite (from O2·− + NO·) reacts directly with PGHS to activate prostaglandin synthesis. Furthermore, removal of O2·− from RAW 267.4 cells that produce NO· and PGHS inhibits prostaglandin biosynthesis to the same extent as NOS inhibitors. This interaction between reactive nitrogen species and PGHS may provide new approaches to the control of inflammation in acute and chronic settings.—Goodwin, D. C., Landino, L. M., Marnett, L. J. Effects of nitric oxide and nitric oxide‐derived species on prostaglandin endoperoxide synthase and prostaglandin biosynthesis. FASEB J. 13, 1121–1136 (1999)

Structural insights into the stereochemistry of the cyclooxygenase reaction.

Cyclooxygenases are bifunctional enzymes that catalyse the first committed step in the synthesis of prostaglandins, thromboxanes and other eicosanoids. The two known cyclooxygenases isoforms share a high degree of amino-acid sequence similarity, structural topology and an identical catalytic mechanism. Cyclooxygenase enzymes catalyse two sequential reactions in spatially distinct, but mechanistically coupled active sites. The initial cyclooxygenase reaction converts arachidonic acid (which is achiral) to prostaglandin G2 (which has five chiral centres). The subsequent peroxidase reaction reduces prostaglandin G2 to prostaglandin H2. Here we report the co-crystal structures of murine apo-cyclooxygenase-2 in complex with arachidonic acid and prostaglandin. These structures suggest the molecular basis for the stereospecificity of prostaglandin G2 synthesis.

Nitric Oxide Trapping of Tyrosyl Radicals Generated during Prostaglandin Endoperoxide Synthase Turnover DETECTION OF THE RADICAL DERIVATIVE OF TYROSINE 385

Tyrosyl radicals have been detected during turnover of prostaglandin endoperoxide H synthase (PGHS), and they are speculated to participate in cyclooxygenase catalysis. Spectroscopic approaches to elucidate the identity of the radicals have not been definitive, so we have attempted to trap the radical(s) with nitric oxide (NO). NO quenched the EPR signal generated by reaction of purified ram seminal vesicle PGHS with arachidonic acid, suggesting that NO coupled with a tyrosyl radical to form inter alianitrosocyclohexadienone. Subsequent formation of nitrotyrosine was detected by Western blotting of PGHS incubated with NO and arachidonic acid or organic hydroperoxides using an antibody against nitrotyrosine. Both arachidonic acid and NO were required to form nitrotyrosine, and tyrosine nitration was blocked by the PGHS inhibitor indomethacin. The presence of superoxide dismutase had no effect on nitration, indicating that peroxynitrite was not the nitrating agent. To identify which tyrosines were nitrated, PGHS was digested with trypsin, and the resulting peptides were separated by high pressure liquid chromatography and monitored with a diode array detector. A single peptide was detected that exhibited a spectrum consistent with the presence of nitrotyrosine. Consistent with Western blotting results, both NO and arachidonic acid were required to observe nitration of this peptide, and its formation was blocked by the PGHS inhibitor indomethacin. Peptide sequencing indicated that the modified residue was tyrosine 385, the source of the putative catalytically active tyrosyl radical.

Catalase in peroxidase clothing: Interdependent cooperation of two cofactors in the catalytic versatility of KatG

Catalase-peroxidase (KatG) is found in eubacteria, archaea, and lower eukaryotae. The enzyme from Mycobacterium tuberculosis has received the greatest attention because of its role in activation of the antitubercular pro-drug isoniazid, and the high frequency with which drug resistance stems from mutations to the katG gene. Generally, the catalase activity of KatGs is striking. It rivals that of typical catalases, enzymes with which KatGs share no structural similarity. Instead, catalatic turnover is accomplished with an active site that bears a strong resemblance to a typical peroxidase (e.g., cytochrome c peroxidase). Yet, KatG is the only member of its superfamily with such capability. It does so using two mutually dependent cofactors: a heme and an entirely unique Met-Tyr-Trp (MYW) covalent adduct. Heme is required to generate the MYW cofactor. The MYW cofactor allows KatG to leverage heme intermediates toward a unique mechanism for H2O2 oxidation. This review evaluates the range of intermediates identified and their connection to the diverse catalytic processes KatG facilitates, including mechanisms of isoniazid activation.

Mesohaem substitution reveals how haem electronic properties can influence the kinetic and catalytic parameters of neuronal NO synthase.

NOSs (NO synthases, EC 1.14.13.39) are haem-thiolate enzymes that catalyse a two-step oxidation of L-arginine to generate NO. The structural and electronic features that regulate their NO synthesis activity are incompletely understood. To investigate how haem electronics govern the catalytic properties of NOS, we utilized a bacterial haem transporter protein to overexpress a mesohaem-containing nNOS (neuronal NOS) and characterized the enzyme using a variety of techniques. Mesohaem-nNOS catalysed NO synthesis and retained a coupled NADPH consumption much like the wild-type enzyme. However, mesohaem-nNOS had a decreased rate of Fe(III) haem reduction and had increased rates for haem-dioxy transformation, Fe(III) haem-NO dissociation and Fe(II) haem-NO reaction with O2. These changes are largely related to the 48 mV decrease in haem midpoint potential that we measured for the bound mesohaem cofactor. Mesohaem nNOS displayed a significantly lower Vmax and KmO2 value for its NO synthesis activity compared with wild-type nNOS. Computer simulation showed that these altered catalytic behaviours of mesohaem-nNOS are consistent with the changes in the kinetic parameters. Taken together, the results of the present study reveal that several key kinetic parameters are sensitive to changes in haem electronics in nNOS, and show how these changes combine to alter its catalytic behaviour.

Plasmodium falciparum Thioredoxin Reductase (PfTrxR) and Its Role as a Target for New Antimalarial Discovery

The growing resistance to current antimalarial drugs is a major concern for global public health. The pressing need for new antimalarials has led to an increase in research focused on the Plasmodium parasites that cause human malaria. Thioredoxin reductase (TrxR), an enzyme needed to maintain redox equilibrium in Plasmodium species, is a promising target for new antimalarials. This review paper provides an overview of the structure and function of TrxR, discusses similarities and differences between the thioredoxin reductases (TrxRs) of different Plasmodium species and the human forms of the enzyme, gives an overview of modeling Plasmodium infections in animals, and suggests the role of Trx functions in antimalarial drug resistance. TrxR of Plasmodium falciparum is a central focus of this paper since it is the only Plasmodium TrxR that has been crystallized and P. falciparum is the species that causes most malaria cases. It is anticipated that the information summarized here will give insight and stimulate new directions in which research might be most beneficial.

Selective Mycobacterium tuberculosis Shikimate Kinase Inhibitors as Potential Antibacterials

Owing to the persistence of tuberculosis (TB) as well as the emergence of multidrug-resistant and extensively drug-resistant (XDR) forms of the disease, the development of new antitubercular drugs is crucial. Developing inhibitors of shikimate kinase (SK) in the shikimate pathway will provide a selective target for antitubercular agents. Many studies have used in silico technology to identify compounds that are anticipated to interact with and inhibit SK. To a much more limited extent, SK inhibition has been evaluated by in vitro methods with purified enzyme. Currently, there are no data on in vivo activity of Mycobacterium tuberculosis shikimate kinase (MtSK) inhibitors available in the literature. In this review, we present a summary of the progress of SK inhibitor discovery and evaluation with particular attention toward development of new antitubercular agents.

Development of an ESI-LC-MS-Based Assay for Kinetic Evaluation of Mycobacterium tuberculosis Shikimate Kinase Activity and Inhibition

A simple and reliable liquid chromatography-mass spectrometry (LC-MS) assay has been developed and validated for the kinetic characterization and evaluation of inhibitors of shikimate kinase from Mycobacterium tuberculosis (MtSK), a potential target for the development of novel antitubercular drugs. This assay is based on the direct determination of the reaction product shikimate-3-phosphate (S3P) using electrospray ionization (ESI) and a quadrupole time-of-flight (Q-TOF) detector. A comparative analysis of the kinetic parameters of MtSK obtained by the LC-MS assay with those obtained by a conventional UV-assay was performed. Kinetic parameters determined by LC-MS were in excellent agreement with those obtained from the UV assay, demonstrating the accuracy, and reliability of this method. The validated assay was successfully applied to the kinetic characterization of a known inhibitor of shikimate kinase; inhibition constants and mode of inhibition were accurately delineated with LC-MS.

Inactivation of myeloperoxidase by benzoic acid hydrazide.

Myeloperoxidase (MPO) is expressed by myeloid cells for the purpose of catalyzing the formation of hypochlorous acid, from chloride ions and reaction with a hydrogen peroxide-charged heme covalently bound to the enzyme. Most peroxidase enzymes both plant and mammalian are inhibited by benzoic acid hydrazide (BAH)-containing compounds, but the mechanism underlying MPO inhibition by BAH compounds is largely unknown. Recently, we reported MPO inhibition by BAH and 4-(trifluoromethyl)-BAH was due to hydrolysis of the ester bond between MPO heavy chain glutamate 242 ((HC)Glu(242)) residue and the heme pyrrole A ring, freeing the heme linked light chain MPO subunit from the larger remaining heavy chain portion. Here we probed the structure and function relationship behind this ester bond cleavage using a panel of BAH analogs to gain insight into the constraints imposed by the MPO active site and channel leading to the buried protoporphyrin IX ring. In addition, we show evidence that destruction of the heme ring does not occur by tracking the heme prosthetic group and provide evidence that the mechanism of hydrolysis follows a potential attack of the (HC)Glu(242) carbonyl leading to a rearrangement causing the release of the vinyl-sulfonium linkage between (HC)Met(243) and the pyrrole A ring.