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 Val 1 catalyzes the first committed step in prostanoid biosynthesis, the bis-dioxygenation of arachidonic acid to form prostaglandin G 2 (1). Two isoforms of PGHS are known, with PGHS-1 generally ascribed housekeeping roles, whereas the strong induction of PGHS-2 by cytokines is believed to be a key part in inflammatory processes (2). Many cyclooxygenase inhibitors have been discovered; the most potent include agents, such as indomethacin, which trigger a time-dependent change in the protein once bound in the cyclooxygenase active site, thus achieving essentially irreversible inhibition without covalent modification of protein or agent (3-5). More recently, a set of time-dependent cyclooxygenase inhibitors with very high selectivity for PGHS-2 has been identified (6 -8). Little is known about the nature of the structural change(s) underlying noncovalent time-dependent cyclooxygenase inhibition of either isoform or about the protein structural features that lead to the remarkable specificity of the PGHS-2 inhibitors.We have constructed a three-dimensional model for human PGHS-2 based on the crystal structure of ovine PGHS-1 (9) and identified Val 509 in PGHS-2 as one of the few residues in the cyclooxygenase active site that is not conserved in PGHS-1. Recombinant human PGHS-2 was expressed with four Val 509 mutations to assess their effects on cyclooxygenase activity and on inhibition by agents specific for PGHS-2. Several of the Val 509 mutations led to a loss of the characteristic time-dependent action of the agents without a large perturbation of substrate or inhibitor binding. The results point to a role for Val 509 in the time-dependent structural transition, which makes these agents such potent and selective inhibitors of human PGHS-2 cyclooxygenase activity. EXPERIMENTAL PROCEDURESMaterials-Heme, dimethyl sulfoxide, and D-tryptophan were from Sigma; Tween 20 was from Pierce; arachidonate was from NuChek Preps, Inc.;[1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]arachidonate (55 mCi/mmol) was from Amersham Corp. Nimesulide was from Cayman Chemical Co. DuP697, SC58125, and NS398 were generous gifts from Drs. Chakk Ramesha (Roche Pharmaceuticals) and Paul J. Marshall (CIBA Pharmaceuticals).Homologous Modeling-The structural model for human PGHS-2 was built from th...

Hydroperoxides induce formation of a tyrosyl radical on Tyr385 in prostaglandin H synthase (PGHS). The Tyr385 radical initiates hydrogen abstraction from arachidonic acid, thereby mechanistically connecting the peroxidase and cyclooxygenase activities. In both PGHS isoforms the tyrosyl radical undergoes a time-dependent transition from a wide doublet to a wide singlet species; pretreatment with cyclooxygenase inhibitors results in a third type of signal, a narrow singlet [Tsai, A.-L.; Kulmacz, R. J. (2000) Prost. Lipid Med. 62, 231-254]. These transitions have been interpreted as resulting from Tyr385 ring rotation, but could also be due to radical migration from Tyr385 to another tyrosine residue. PATHWAYS analysis of PGHS crystal structures identified four tyrosine residues with favorable predicted electronic coupling: residues 148, 348, 404, and 504 (ovine PGHS-1 numbering). We expressed recombinant PGHS-2 proteins containing single Tyr --> Phe mutations at the target residues, a quadruple mutant with all four tyrosines mutated, and a quintuple mutant, which also contains a Y385F mutation. All mutants bind heme and display appreciable peroxidase activity, and with the exception of the quintuple mutant, all retain cyclooxygenase activity, indicating that neither of the active sites is significantly perturbed. Reaction of the Y148F, Y348F, and Y404F mutants with EtOOH generates a wide singlet EPR signal similar to that of native PGHS-2. However, reaction of the Y504F and the quadruple mutants with peroxide yields persistent wide doublets, and the quintuple mutant is EPR silent. Nimesulide pretreatment of Y504F and the quadruple mutant results in an abnormally small amount of wide doublet signal, with no narrow singlet being formed. Therefore, the formation of an alternative tyrosine radical on Tyr504 probably accounts for the transition from a wide doublet to a wide singlet in native PGHS-2 and for formation of a narrow singlet in complexes of PGHS-2 with cyclooxygenase inhibitors.

The mineralization process associated with the conversion of predentin to dentin is believed to be initiated and controlled by a set of acidic regulatory noncollagenous proteins (NCPs) which include phosphophoryn, the major NCP in dentin. Phosphophoryn binds tightly to collagen and is believed to initiate the formation of apatite crystals which play a central role in the mineralization process. During the process of analyzing the 3 end of an odontoblast-specific cDNA which codes for dentin sialoprotein (Ritchie, H. H., Hou, H., Veis, A., and Butler, W. T. (1994) J. Biol. Chem. 269, 3698 -3702), we discovered a 801-base pair open reading frame. This downstream open reading frame encodes a putative leader sequence and a very acidic mature protein sequence having a deduced amino acid composition containing high percentages of both Ser (43%) and Asp (31%) residues which closely coincides with the amino acid composition of phosphophoryns from human, bovine, rat, and rabbit (i.e. Asp (30 -40%) and Ser (38 -50%)). This newly identified cDNA therefore encodes a protein with characteristics similar to phosphophoryn. Here we present the cDNA sequence, the deduced amino acid sequence, and the prospective Ser residue-specific casein kinase I and II phosphorylation sites for this putative phosphophoryn.The calcification process that accompanies the transition of predentin to dentin is poorly understood, due in part to the difficulties in isolating and characterizing unique sets of extracellular matrix molecules that contribute to this complex process (1-4). Phosphophoryn, the most abundant noncollagenous protein in dentin, is secreted by odontoblasts through odontoblastic processes and appears at the mineralization front within a short time after labeling with [ 33 P]phosphate (5, 6). Phosphophoryn is known to bind large amounts of calcium with a relatively high affinity (7) and to then form an insoluble aggregate in the presence of Mg 2ϩ and Ca 2ϩ (8). Because of its affinity for calcium, phosphophoryn may concentrate these ions and participate in the formation of apatite crystals. For example, Linde and co-workers (9) have demonstrated that when phosphophoryn is immobilized on a stable support and incubated in physiological solutions of calcium and phosphate, phosphophoryn induced the formation of hydroxyapatite (HAP).1 Studies by the same group (10) and by Boskey et al. (11) also suggested a dual role for phosphophoryn as both an initiator of HAP formation at low phosphophoryn concentrations and as an inhibitor of HAP formation at higher phosphophoryn concentrations.Phosphophoryn is also believed to have a specific affinity for collagen (2, 12, 13) which comprises as much as 80% of the protein in dentin. Furthermore, phosphophoryn was found to be specifically associated with the "e" band of collagen (14). This site-specific protein-protein interaction, coupled with phosphophoryn's ability to initiate or inhibit HAP formation when calcium is present, has lead to the currently accepted view that phosphophoryn plays a centra...

Prostacyclin is a potent mediator of vasodilation and anti-platelet aggregation. It is synthesized from prostaglandin H(2) by prostacyclin synthase (PGIS), a member of Family 8 in the cytochrome P450 superfamily. Unlike most P450s, which require exogenous reducing equivalents and an oxygen molecule for mono-oxygenation, PGIS catalyzes an isomerization with an initial step of endoperoxide bond cleavage of prostaglandin H(2) (PGH(2)). The low abundance of PGIS in natural tissues necessitates heterologous expression for studies of structure/function relationships and reaction mechanism. We report here a high-yield prokaryotic system for expression of enzymatically active human PGIS. The PGIS cDNA is modified by replacing the hydrophobic amino-terminal sequence with the more hydrophilic amino-terminal sequence from P450 2C5 and by adding a four-histidine tag at the carboxyl terminus. The resulting recombinant PGIS associates with host cell membranes and was purified to electrophoretic homogeneity by nickel affinity, hydroxyapatite and CM Sepharose column chromatography. The recombinant PGIS, with a heme:protein ratio of 0.9:1, catalyzes prostacyclin formation at a K(m) of 13.3 muM PGH(2) and a V(max) of 980 per min. The dithionite-reduced PGIS binds CO with an on-rate of 5.6 x 10(5) M(-1) s(-1) and an off-rate of 15 s(-1). The ferrous-CO complex of PGIS is very short-lived and decays at a rate of 0.7 s(-1). Spectral binding assays showed that imidazole binds weakly to PGIS (K(d) approximately 0.5 mM,) but clotrimazole, a bulky and rigid imidazole derivative, binds strongly (K(d) approximately 1 microM). The transient nature of the CO complex and the weak imidazole binding seem to support an earlier proposal that PGIS active site has a limited space, but the tight binding of clotrimazole argues against this view. It appears that the heme distal pocket of PGIS is fairly adaptable to ligands of various structures. UV-visible absorption, magnetic circular dichroism and electron paramagnetic resonance spectra indicate that PGIS has a typical low-spin heme with a hydrophobic active site. PGIS catalyzes homolytic scission of the peroxide bond of a test substrate, 10-hydroperoxyoctadeca-8,12-dienoic acid, accompanied by formation of a heme intermediate with a Compound II-like optical spectrum.

Characterization of the redox properties of endothelial nitric-oxide synthase (eNOS) is fundamental to understanding the complicated reaction mechanism of this important enzyme participating in cardiovascular function. Yeast overexpression of both the oxygenase and reductase domains of human eNOS, i.e. eNOS ox and eNOS red , has been established to accomplish this goal. UV-visible and electron paramagnetic resonance (EPR) spectral characterization for the resting eNOS ox and its complexes with various ligands indicated a standard NOS heme structure as a thiolate hemeprotein. Two low spin imidazole heme complexes but not the isolated eNOS ox were resolved by EPR indicating slight difference in heme geometry of the dimeric eNOS ox domain. Stoichiometric titration of eNOS ox demonstrated that the heme has a capacity for a reducing equivalent of 1-1.5. Additional 1.5-2.5 reducing equivalents were consumed before heme reduction occurred indicating the presence of other unknown high potential redox centers. There is no indication for additional metal centers that could explain this extra electron capacity of eNOS ox . Ferrous eNOS ox , in the presence of L-arginine, is fully functional in forming the tetrahydrobiopterin radical upon mixing with oxygen as demonstrated by rapidfreeze EPR measurements. Calmodulin binds eNOS red at 1:1 stoichiometry and high affinity. Stoichiometric titration and computer simulation enabled the determination for three redox potential separations between the four half-reactions of FMN and FAD. The extinction coefficient could also be resolved for each flavin for its semiquinone, oxidized, and reduced forms at multiple wavelengths. This first redox characterization on both eNOS domains by stoichiometric titration and the generation of a high quality EPR spectrum for the BH 4 radical intermediate illustrated the usefulness of these tools in future detailed investigations into the reaction mechanism of eNOS. Nitric-oxide synthase (NOS)1 is an uncommon self-sufficient P450-like enzyme catalyzing nitric oxide (NO) biosynthesis from L-arginine (1-4). There are three mammalian NOS isozymes: the constitutive neuronal NOS (nNOS) and endothelial NOS (eNOS) require calmodulin for enzyme activity, whereas the inducible NOS (iNOS) contains tightly bound calmodulin (1-4). All three isozymes have a common bi-domain structure with the reductase domain containing FAD, FMN, and NADPH binding sites, and the oxygenase domain harboring the heme center and binding sites for L-arginine and tetrahydrobiopterin (BH 4 ) (1-4). The main function of the reductase domain is to provide reducing equivalents to the heme center in the oxygenase domain where the key chemistry of L-arginine conversion occurs. Three substrates and four products are involved in NOS catalysis. The overall reaction is a complicated five-electron oxidation of the key guanidine nitrogen plus three additional electrons from NADPH to reduce two molecules of oxygen to water and form the L-citrulline and nitric oxide. Several x-ray crystallographic s...