Prostaglandins are synthesized by prostaglandin H synthase (PGHS) 1 and 2. PGHS2 is regulated through inducible expression. We report here the regulation of PGHS1 activity by substrate-dependent cooperative activation. The cooperativity is characterized by a Hill coefficient of 1.29 ؎ 0.06, a curved Eadie-Scatchard plot, and activation by low concentrations of competitive inhibitors. The activation also appears to induce a conformational change in the cyclooxygenase site. The cooperativity produces a 2-4-fold greater rate of PGHS2-dependent prostaglandin formation compared with PGHS1-dependent prostaglandin formation at arachidonic acid concentrations below 0.5 M. A consequence of the PGHS1 cooperativity is that the affinity of many cyclooxygenase inhibitors for PGHS1 decreases in parallel to the activation by arachidonic acid. In contrast, the affinity of these inhibitors for PGHS2 is unaffected by the changes in arachidonic acid concentration. This results in a dramatic difference in PGHS2/PGHS1 selectivity at different arachidonic acid concentrations. Prostaglandin H synthase (PGHS)1 oxidizes arachidonic acid to prostaglandin H 2 , which is the precursor of all other prostaglandins. Two forms of PGHS are known: constitutively expressed PGHS1 and inducible PGHS2. PGHS1 is considered to be the housekeeping enzyme, whereas PGHS2 is induced in response to cellular stress and is thought to play a role in inflammation (1, 2). Aspirin and other nonsteroidal anti-inflammatory drugs inhibit both enzymes (3). Chronic use of these drugs causes stomach bleeding and kidney failure. These dangerous side effects are thought to be due to inhibition of PGHS1-dependent prostaglandin synthesis and not inhibition of PGHS2 (4). Recently, PGHS2 expression has been shown to be associated with colon cancer (5, 6) and Alzheimer's disease (7,8). Accordingly, extensive biological and pharmaceutical research has focused on factors that differentiate between these two routes of prostaglandin biosynthesis.Accumulating biological evidence points to a functional differentiation in the formation of prostaglandins by PGHS1 and PGHS2. Both enzymes are located in the endoplasmic reticulum and the nuclear envelope with PGHS2 somewhat more concentrated in the nuclear envelope (9). Murakami et al. (10,11) showed that both enzymes function independently by coupling different stimulus-initiated pathways to prostaglandin D 2 generation from endogenous arachidonic acid. Reddy and Herschman (12, 13) have proposed at least two independent pathways for prostaglandin synthesis: a secretory phospholipase A 2 -mediated, PGHS1-dependent transcellular pathway and an intracellular cytosolic phospholipase A 2 -mediated, PGHS2-dependent pathway. This is consistent with the hypothesis that PGHS1 prefers exogenous arachidonic acid, and PGHS2 prefers endogenous arachidonic acid (12). Chulada and co-workers (14) quantitated the differences in the rate of prostaglandin formation by PGHS1 and PGHS2 in mouse embryonic fibroblasts and Chinese hamster ovary cells with st...

A small molecule inhibitor of NF-B-dependent cytokine expression was discovered that blocked tumor necrosis factor (TNF) ␣-induced IB␣ degradation in MM6 cells but not the degradation of ␤-catenin in Jurkat cells. Ro106-9920 blocked lipopolysaccharide (LPS)-dependent expression of TNF␣, interleukin-1␤, and interleukin-6 in fresh human peripheral blood mononuclear cells with IC 50 values below 1 M. Ro106-9920 also blocked TNF␣ production in a dose-dependent manner following oral administration in two acute models of inflammation (air pouch and LPS challenge). Ro106-9920 was observed to inhibit an ubiquitination activity that does not require ␤TRCP but associates with IB␣ and will ubiquitinate IB␣ S32E,S36E (IB␣ee) specifically at lysine 21 or 22. Ro106-9920 was identified in a cell-free system as a time-dependent inhibitor of IB␣ee ubiquitination with an IC 50 value of 2.3 ؎ 0.09 M. The ubiquitin E3 ligase activity is inhibited by cysteine-alkylating reagents, supported by E2UBCH7, and requires cIAP2 or a cIAP2-associated protein for activity. These activities are inconsistent with what has been reported for SCF ␤TRCP , the putative E3 for IB␣ ubiquitination. Ro106-9920 was observed to be selective for IB␣ee ubiquitination over the ubiquitin-activating enzyme (E1), E2UBCH7, nonspecific ubiquitination of cellular proteins, and 97 other molecular targets. We propose that Ro106-9920 selectively inhibits an uncharacterized but essential ubiquitination activity associated with LPSand TNF␣-induced IB␣ degradation and NF-B activation.

CYP17 catalyzes the cleavage of the C-17 side chain of progesterone to form androstenedione. The two-step reaction involves an initial 17 alpha-hydroxylation catalyzed by oxene chemistry followed by cleavage of the C-17 side chain. We have recently shown that C-17 side-chain cleavage may involve the rearrangement of a peroxy intermediate via a Baeyer-Villiger rearrangement [Mak, A. Y., & Swinney, D. C. (1992) J. Am. Chem. Soc. 114, 8309]. Accordingly, CYP17 is proposed to catalyze oxidations via both oxene and peroxide chemistry. This study was initiated to investigate the possibility that protons may play a determining role in differentiating between the oxene and peroxide chemistries associated with product formation. The pL dependence of the deuterium solvent isotope effects associated with progesterone oxidation to 17 alpha-hydroxyprogesterone and 17-O-acetyltestosterone and 17 alpha-hydroxyprogesterone oxidation to androstenedione was determined in microsomes from pig testes. The formation of 17 alpha-hydroxyprogesterone is assumed to occur via oxene chemistry and the formation of 17-O-acetyltestosterone and androstenedione by peroxide chemistry. The initial rate of progesterone oxidation to 17 alpha-hydroxyprogesterone was associated with a pL-independent inverse solvent isotope effect (Hk/Dk = 0.75-0.95, in 30% DOD), whereas the rate of oxidation to 17-O-acetyltestosterone was associated with a pL-independent positive solvent isotope effect in the presence of 30% DOD (Hk/Dk approximately 2). In contrast, DOD inhibited the formation of androstenedione from 17 alpha-hydroxyprogesterone in a noncompetitive, pL-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Prostaglandin H synthases (PGHSs) catalyze the conversion of arachidonic acid to prostaglandins. In this report, we describe the effect of a PGHS2 Y355F mutation on the dynamics of PGHS2 catalysis and inhibition. Tyr 355 is part of a hydrogen-bonding network located at the entrance to the cyclooxygenase active site. The Y355F mutant exhibited allosteric activation kinetics in the presence of arachidonic acid that was defined by a curved Eadie-Scatchard plot and a Hill coefficient of 1.36 ؎ 0.05. Arachidonic acid-induced allosteric activation has not been directly observed with wild type PGHS2. The mutation also decreased the observed timedependent inhibition by indomethacin, flurbiprofen, RS-57067, and SC-57666. Detailed kinetic analysis showed that the Y355F mutation decreased the transition state energy associated with slow-binding inhibition (EI ‡) relative to the energy associated with catalysis (ES ‡) by 1.33, 0.67, and 1.06 kcal/mol, respectively, for indomethacin, flurbiprofen, and RS-57067. These observations show Tyr 355 to be involved in the molecular mechanism of time-dependent inhibition. We interpret these results to indicate that slow binding inhibitors and the Y355F mutant slow the rate and unmask intrinsic, dynamic events associated with product formation. We hypothesize that the dynamic events are the equilibrium between relaxed and tightened organizations of the hydrogen-bonding network at the entrance to the cyclooxygenase active site. It is these rearrangements that control the rate of substrate binding and ultimately the rate of prostaglandin formation.Prostaglandins are formed from arachidonic acid by constitutive prostaglandin H synthase 1 (PGHS1) 1 and inducible prostaglandin H synthase 2 (PGHS2) (1). They are important cellular mediators of many biological functions, including inflammation, pyresis, and algesia (2, 3). Recent evidence suggests that prostaglandins formed by PGHS2 mediate inflammation (3, 4). PGHS2 is also implicated in the pathology of Alzheimer's disease and colon cancer (5-7).The latest methodologies in structure-based drug discovery are being used to identify PGHS2-selective medicines. Inhibitor-bound structures of PGHS1 and PGHS2 have been solved (8 -11). The structures and additional mutagenesis data have identified a number of important features (12-17) (Fig. 1). The inhibitors bind in a long channel whose entrance is flanked by three residues capable of creating a hydrogen-bonding network, Arg 120 , 2 Glu 524 , and Tyr 355 . Arg 120 is required for binding the carboxylic acid moiety of fatty acid substrates and nonsteroidal anti-inflammatory drugs (12, 13). Tyr 355 is proposed to be a determinant of specificity in the 2-phenylproprionic class of inhibitors; inhibition of the PGHS1 phenylalanine mutant by ibuprofen produced a change in the stereochemical specificity but not potency (13). The channel ends at residue Tyr 385 , a residue required for cyclooxygenase catalytic activity (14). The channel is bordered by Ser 530 , the site of aspirin acetylation and a side ...

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