Richard J. Kulmacz

Comparison of Hydroperoxide Initiator Requirements for the Cyclooxygenase Activities of Prostaglandin H Synthase-1 and −2

Two isoforms of prostaglandin H synthase have been described: isoform-1 (PGHS-1), which is ascribed a role in basal or housekeeping prostaglandin synthesis; and isoform-2 (PGHS-2), which has been found to be strongly inducible in many tissues and has been associated with inflammatory processes. Recent observations have indicated that cyclooxygenase catalysis by the two isoforms can be differentially regulated when both are present simultaneously (Reddy, S. T., and Herschman, H. R.(1994) J. Biol. Chem. 269, 15473-15480). The requirement of the cyclooxygenase for hydroperoxide initiator has been proposed as an important limit on cellular prostaglandin synthesis (Marshall, P. J., Kulmacz, R. J., and Lands, W. E. M.(1987) J. Biol. Chem. 262, 3510-3517). To compare the levels of hydroperoxide required for cyclooxygenase initiation in the two PGHS isoforms, we have examined the ability of a hydroperoxide scavenger, glutathione peroxidase, to suppress the cyclooxygenase activity of purified preparations of human PGHS-2, ovine PGHS-2, and ovine PGHS-1. Half-maximal prostaglandin synthetic activity was found to require a much lower hydroperoxide level with human PGHS-2 (2.3 nM) and ovine PGHS-2 (2.2 nM) than with ovine PGHS-1 (21 nM). Similar results were obtained when cyclooxygenase activity was monitored by chromatographic analyses of radiolabeled arachidonate metabolites or with oxygen electrode measurements. Mixing four parts of ovine PGHS-1 with one part of human PGHS-2 did not markedly change the sensitivity of the overall cyclooxygenase activity to inhibition by glutathione peroxidase, indicating that the PGHS-1 activity was not easily initiated by PGHS-2 activity in the same vessel. Effective catalysis by PGHS-2 can thus proceed at hydroperoxide levels too low to sustain appreciable catalysis by PGHS-1. This difference in catalytic characteristics provides a biochemical mechanism for differential control of prostaglandin synthesis by the two PGHS isoforms, even when both are present in the same intracellular compartment.

Role of Val509 in Time-dependent Inhibition of Human Prostaglandin H Synthase-2 Cyclooxygenase Activity by Isoform-selective Agents

Prostaglandin H synthase (PGHS), a key enzyme in prostanoid biosynthesis, exists as two isoforms. PGHS-1 is considered a basal enzyme; PGHS-2 is associated with inflammation and cell proliferation. A number of highly selective inhibitors for PGHS-2 cyclooxygenase activity are known. Inhibition by these agents involves an initial reversible binding, followed by a time-dependent transition to a much higher affinity enzyme-inhibitor complex, making these agents potent and poorly reversible PGHS-2 inhibitors. To investigate the PGHS-2 structural features that influence the time-dependent action of the selective inhibitors, we have constructed a three-dimensional model of human PGHS-2 by homologous modeling. Examination of the PGHS-2 model identified Val509 as a cyclooxygenase active site residue, that was not conserved in PGHS-1. Recombinant human PGHS-2 with Val509 mutated to either Ile (the corresponding residue in PGHS-1), Ala, Glu, or Lys was expressed by transient transfection of COS-1 cells to evaluate the effects of the mutations on cyclooxygenase activity and on inhibition by four agents reported to be selective for PGHS-2 (NS398, nimesulide, DuP697, and SC58125). All the recombinant proteins were of the expected mass. The mutants exhibited 45-210% of wild-type cyclooxygenase activity, with Km values for arachidonate of 2.1-7.6 μM (wild-type PGHS-2, 3.8 μM), indicating that changes in position 509 had modest effects on cyclooxygenase catalysis. Each of the agents inhibited wild-type PGHS-2 in a time-dependent fashion, and all but nimesulide did the same for the V509A mutant. In contrast, the V509E and V509I PGHS-2 mutants, like recombinant human PGHS-1, did not show time-dependent inhibition with any of the agents, and the V509K mutant responded in a time-dependent manner only to DuP697. Reversible inhibition was still observed with Val509 mutants that did not show time-dependent inhibition. Thus, the side chain structure at position 509 markedly influenced the ability of PGHS-2 to undergo the time-dependent transition without removing inhibitor or substrate binding. These results indicate that Val509 in PGHS-2 has a major role in the structural transition that underlies time-dependent inhibition by the isoform-selective agents.

A Mechanistic Study of Self-inactivation of the Peroxidase Activity in Prostaglandin H Synthase-1

Prostaglandin H synthase (PGHS) is a self-activating and self-inactivating enzyme. Both the peroxidase and cyclooxygenase activities have a limited number of catalytic turnovers. Sequential stopped-flow measurements were used to analyze the kinetics of PGHS-1 peroxidase self-inactivation during reaction with several different hydroperoxides. The inactivation followed single exponential kinetics, with a first-order rate constant of 0.2–0.5 s−1 at 24 °C. This rate was independent of the peroxide species and concentration used, strongly suggesting that the self-inactivation process originates after formation of Compound I and probably with Intermediate II, which contains an oxyferryl heme and a tyrosyl radical. Kinetic scan and rapid scan experiments were used to monitor the heme changes during the inactivation process. The results from both experiments converged to a simple, linear, two-step mechanism in which Intermediate II is first converted in a faster step (0.5–2 s−1) to a new compound, Intermediate III, which undergoes a subsequent slower (0.01–0.05 s−1) transition to a terminal species. Rapid-quench and high pressure liquid chromatography analysis indicated that Intermediate III likely retains an intact heme group that is not covalently linked with the PGHS-1 protein.

Comparison of the peroxidase reaction kinetics of prostaglandin H synthase-1 and -2.

Prostaglandin H synthase isoforms 1 and 2 (PGHS-1 and -2) each have a peroxidase activity and also a cyclooxygenase activity that requires initiation by hydroperoxide. The hydroperoxide initiator requirement for PGHS-2 cyclooxygenase is about 10-fold lower than for PGHS-1 cyclooxygenase, and this difference may contribute to the distinct control of cellular prostanoid synthesis by the two isoforms. We compared the kinetics of the initial peroxidase steps in PGHS-1 and -2 to quantify mechanistic differences between the isoforms that might contribute to the difference in cyclooxygenase initiation efficiency. The kinetics of formation of Intermediate I (an Fe(IV) species with a porphyrin free radical) and Intermediate II (an Fe(IV) species with a tyrosyl free radical, thought to be the crucial oxidant in cyclooxygenase catalysis) were monitored at 4°c by stopped flow spectrophotometry with several hydroperoxides as substrate. With 15-hydroperoxyeicosatetraenoic acid, the rate constant for Intermediate I formation (k 1) was 2.3 × 107 m −1 s−1 for PGHS-1 and 2.5 × 107 m −1s−1 for PGHS-2, indicating that the isoforms have similar initial reactivity with this lipid hydroperoxide. For PGHS-1, the rate of conversion of Intermediate I to Intermediate II (k 2) became the limiting factor when the hydroperoxide level was increased, indicating a rate constant of 102–103 s−1 for the generation of the active cyclooxygenase species. For PGHS-2, however, the transition between Intermediates I and II was not rate-limiting even at the highest hydroperoxide concentrations tested, indicating that thek 2 value for PGHS-2 was much greater than that for PGHS-1. Computer modelling predicted that faster formation of the active cyclooxygenase species (Intermediate II) or increased stability of the active species increases the resistance of the cyclooxygenase to inhibition by the intracellular hydroperoxide scavenger, glutathione peroxidase. Kinetic differences between the PGHS isoforms in forming or stabilizing the active cyclooxygenase species can thus contribute to the difference in the regulation of their cellular activities.

Rapid Kinetics of Tyrosyl Radical Formation and Heme Redox State Changes in Prostaglandin H Synthase-1 and -2

Hydroperoxide-induced tyrosyl radicals are putative intermediates in cyclooxygenase catalysis by prostaglandin H synthase (PGHS)-1 and -2. Rapid-freeze EPR and stopped-flow were used to characterize tyrosyl radical kinetics in PGHS-1 and -2 reacted with ethyl hydrogen peroxide. In PGHS-1, a wide doublet tyrosyl radical (34–35 G) was formed by 4 ms, followed by transition to a wide singlet (33–34 G); changes in total radical intensity paralleled those of Intermediate II absorbance during both formation and decay phases. In PGHS-2, some wide doublet (30 G) was present at early time points, but transition to wide singlet (29 G) was complete by 50 ms. In contrast to PGHS-1, only the formation kinetics of the PGHS-2 tyrosyl radical matched the Intermediate II absorbance kinetics. Indomethacin-treated PGHS-1 and nimesulide-treated PGHS-2 rapidly formed narrow singlet EPR (25–26 G in PGHS-1; 21 G in PGHS-2), and the same line shapes persisted throughout the reactions. Radical intensity paralleled Intermediate II absorbance throughout the indomethacin-treated PGHS-1 reaction. For nimesulide-treated PGHS-2, radical formed in concert with Intermediate II, but later persisted while Intermediate II relaxed. These results substantiate the kinetic competence of a tyrosyl radical as the catalytic intermediate for both PGHS isoforms and also indicate that the heme redox state becomes uncoupled from the tyrosyl radical in PGHS-2.

Cellular regulation of prostaglandin H synthase catalysis

Prostanoids are a group of potent bioactive lipids produced by oxygenation of arachidonate or one of several related polyunsaturated fatty acids. Cellular prostaglandin biosynthesis is tightly regulated, with a large part of the control exerted at the level of cyclooxygenase catalysis by prostaglandin H synthase (PGHS). The two known isoforms of PGHS have been assigned distinct pathophysiological functions, and their cyclooxygenase activities are subject to differential cellular control. This review considers the contributions to cellular catalytic control of the two PGHS isoforms by intracellular compartmentation, accessory proteins, arachidonate levels, and availability of hydroperoxide activator.

Prostaglandin H synthase: perturbation of the tyrosyl radical as a probe of anticyclooxygenase agents.

EPR spectroscopy was used to study the effects of various nonsteroidal anti-inflammatory agents on the peroxidase-related tyrosyl radical present in prostaglandin H synthase (prostaglandin endoperoxide synthase; EC 1.14.99.1). Two types of perturbation of the tyrosyl radical by these anticyclooxygenase agents were observed. In the first case, aspirin, indomethacin, ibuprofen, (S)-flurbiprofen, and (S)-naproxen converted the doublet tyrosyl EPR signal seen on reaction of the uninhibited enzyme with ethyl hydroperoxide to a singlet bearing additional partially resolved hyperfine splittings. These compounds also decreased the maximum amount of radical generated, but they did not change the kinetics of formation and decay of the tyrosyl radical. In the second case, acetaminophen and three fenamate analogs (meclofenamate, flufenamate, and mefenamate) did not perturb the EPR line shape observed after reaction with hydroperoxide but did cause a more rapid decay of the tyrosine radical species. It would appear that, despite considerable variation in structure, the nonsteroidal anti-inflammatory agents may inhibit the cyclooxygenase activity of the synthase by two basic mechanisms.

Expression, Purification, and Spectroscopic Characterization of Human Thromboxane Synthase

Thromboxane A2(TXA2) is a potent inducer of vasoconstriction and platelet aggregation. Large scale expression of TXA2 synthase (TXAS) is very useful for studies of the reaction mechanism, structural/functional relationships, and drug interactions. We report here a heterologous system for overexpression of human TXAS. The TXAS cDNA was modified by replacing the sequence encoding the first 28 amino acid residues with a CYP17 amino-terminal sequence and by adding a polyhistidine tag sequence prior to the stop codon; the cDNA was inserted into the pCW vector and co-expressed with chaperonins groES and groEL in Escherichia coli. The resulting recombinant protein was purified to electrophoretic homogeneity by affinity, ion exchange, and hydrophobic chromatography. UV-visible absorbance (UV-Vis), magnetic circular dichroism (MCD), and electron paramagnetic resonance (EPR) spectra indicate that TXAS has a typical low spin cytochrome P450 heme with an oxygen-based distal ligand. The UV-Vis and EPR spectra of recombinant TXAS were essentially identical to those of TXAS isolated from human platelets, except that a more homogenous EPR spectrum was observed for the recombinant TXAS. The recombinant protein had a heme:protein molar ratio of 0.7:1 and a specific activity of 12 μmol of TXA2/min/mg of protein at 23 °C. Furthermore, it catalyzed formation of TXA2, 12-hydroxy-5,8,10-heptadecatrienoic acid, and malondialdehyde in a molar ratio of 0.94:1.0:0.93. Spectral binding titrations showed that bulky heme ligands such as clotrimazole bound strongly to TXAS (K d ∼0.5 μm), indicating ample space at the distal face of the heme iron. Analysis of MCD and EPR spectra showed that TXAS was a typical low spin hemoprotein with a proximal thiolate ligand and had a very hydrophobic distal ligand binding domain.

Identification of Thromboxane A2 Synthase Active Site Residues by Molecular Modeling-guided Site-directed Mutagenesis

Human thromboxane A2 synthase (TXAS) exhibits spectral characteristics of cytochrome P450 but lacks monooxygenase activity. Its distinctive amino acid sequence makes TXAS the sole member of family 5 in the P450 superfamily. To better understand the structure-function relationship of this unusual P450, we have recently constructed a three-dimensional model for TXAS using P450BM-3 as the template (Ruan, K.-H., Milfeld, K., Kulmacz, R. J., and Wu, K. K. (1994) Protein Eng. 7, 1345-1551) and have identified a potential active site region. The catalytic roles of several putative active site residues were evaluated using selectively mutated recombinant TXAS expressed in COS-1 cells. Mutation of Ala-408 to Glu or Arg-413 to Gly led to a complete loss of enzyme activity despite expression of mutant protein levels equivalent to that of the wild-type TXAS. Mutation of Ala-408 to Gly or Leu retained the enzyme activity at levels of 30 or 40%, respectively. This suggests that Ala-408 provides a hydrophobic environment for substrate binding. Mutation of Arg-413 to Lys or Gln completely abolished the enzyme activity, indicating that this residue is essential to catalytic activity and supports its identification as an active site residue. Mutation of Arg-410 to Gly or Glu-433 to Ala resulted in >50% reduction in the enzyme activity without appreciably altering mutant protein expression, consistent with a more subtle effect of these residues on TXAS catalytic efficiency. Mutation of residues predicted to be involved in binding the heme prosthetic group, including the heme thiolate ligand Cys-480, Arg-478, Phe-127, and Asn-110, each markedly reduced the expressed protein level and abolished enzyme activity. This suggests that proper heme binding is important to synthesis or stability of recombinant TXAS. Mutation of Ile-346, which corresponds to P450cam-Thr-252, an essential amino acid involved in dioxygen bond scission, to Thr increased the enzymatic activity by 40%, suggesting that oxygen bond cleavage is not a rate-limiting step in thromboxane A2 biosynthesis. The present results from site-directed mutagenesis support the overall structure of the TXAS active site predicted by homology modeling and have allowed refinement of the position of bound substrate.

Peroxide Tone in Eicosanoid Signaling

The close association of lipid hydroperoxides (ROOH) and hydrogen peroxide (HOOH) with inflammatory/proliferative processes sets the context for examining whether those peroxides are intermediates without which the processes could not occur or are simply extraneous waste products. The peroxides and the processes are so intertwined that “oxidative stress” and signal transductions paradoxically seem at times to change their roles, as a cause produces an effect that then becomes a cause for further effects. The need to intervene successfully in a wide range of inflammatory/proliferative disorders makes it important to develop a functional understanding of the degree to which ambient levels of hydroperoxides influence those disorders. Those ambient levels constitute the peroxide tone discussed in this chapter.