The dynamics of prostaglandin H synthases. Studies with prostaglandin h synthase 2 Y355F unmask mechanisms of time-dependent inhibition and allosteric activation.

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. Tyr355 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 time-dependent 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 (EIdouble dagger) relative to the energy associated with catalysis (ESdouble dagger) by 1.33, 0.67, and 1.06 kcal/mol, respectively, for indomethacin, flurbiprofen, and RS-57067. These observations show Tyr355 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.

The kinetic factors that determine the affinity and selectivity for slow binding inhibition of human prostaglandin H synthase 1 and 2 by indomethacin and flurbiprofen.

We present here for the first time a method for determining the rate constants associated with slow binding inhibition of prostaglandin H synthase (PGHS). The rate constants were determined by a method using initial steady-state conditions, which minimize the impact of catalytic autoinactivation of the enzyme. The currently available methods for determining the kinetic constants associated with slow binding enzyme inhibition do not distinguish between rate decreases due to enzyme inhibition or due to autoinactivation of the enzyme. A mathematical model was derived assuming a rapid reversible formation of an initial enzyme-inhibitor complex (EI) followed by a slow reversible formation of a second enzyme-inhibitor complex (EI*). The two enzyme inhibitor complexes are assumed to be in slow equilibrium. This method was used to evaluate the kinetic parameters associated with the binding and selectivity of the nonsteroidal antinflammatory drugs (NSAIDs), flurbiprofen and indomethacin. The KI values associated with the formation of the first reversible complex (EI) for flurbiprofen with PGHS1 and PGHS2 were 0.53 +/- 0. 06 and 0.61 +/- 0.08 microM, respectively; the rate constants for the forward isomerization, k2, into the second reversible complex (EI*) were 0.97 +/- 0.99 and 0.11 +/- 0.01 s-1, respectively, and rates of the reverse isomerization from EI*, k-2, were 0.031 +/- 0.004 and 0.0082 +/- 0.0008 s-1, respectively. Indomethacin was estimated to form the EI complex with the same affinity for both PGHS1 and PGHS2, 10.0 +/- 2.8 microM and 11.2 +/- 2.0 microM, respectively, and dissociate from EI* at approximately the same rate 0.0011 +/- 0.0002 s-1 and 0.0031 +/- 0.0003 s-1, respectively. However, the rate of isomerization into EI* from EI was much greater for PGHS1 than PGHS2, 0.33 +/- 0.08 s-1 as compared with 0.034 +/- 0.004 s-1. These results show that the overall affinity for the inhibition of PGHS1 versus PGHS2 was 30-fold greater for indomethacin (KI* = 0.032 +/- 0.005 and 1.02 +/- 0.08 microM, respectively) and 3-fold greater for flurbiprofen (KI* = 0.017 +/- 0.002 and 0.045 +/- 0.004 microM, respectively). The results also show that for both PGHS1 and PGHS2, flurbiprofen was bound tighter to the initial EI complex than indomethacin; however, the rate of dissociation from EI* was slower for indomethacin than flurbiprofen. The rate of the forward isomerization to EI* is primarily responsible for the selectivity of both NSAIDs for PGHS1. This analysis shows the quantitative importance of the different kinetic parameters upon the overall binding affinity of these NSAIDs and should greatly assist in our understanding of the structural interactions that promote enzyme-inhibitor binding.

Cloning and functional expression of the cDNA encoding rat lanosterol 14-alpha demethylase.

Lanosterol 14 alpha-demethylase (LDM) is a cytochrome P-450 enzyme in the biosynthetic pathway of cholesterol. As such, it represents a target for cholesterol-lowering drugs. Rat LDM (rLDM) has been purified from the livers of rats treated with cholestyramine. The purified protein was used to generate tryptic fragments which were then sequenced. The amino acid (aa) sequences were used to design oligodeoxyribonucleotide primers and a DNA fragment was generated by RT-PCR to probe a phagemid library. A clone encoding rLDM was isolated from the livers of cholestyramine-treated rats. The clone contains an open reading frame encoding a polypeptide of 486 aa and a predicted molecular mass of 55 045 Da. The deduced aa sequence shows a high degree of identity to the yeast LDM sequences, as well as sequences which match typical P-450 sequence motifs. When produced in a baculovirus/insect cell culture system, LDM activity was detected and inhibited by the specific inhibitor azalanstat with an IC50 value of less than 2 nM. The isolation of this full-length coding sequence should facilitate research into understanding the direct and indirect effects of LDM in the regulation of cholesterol biosynthesis and the search for cholesterol-lowering drugs.

Selective inhibition of mammalian lanosterol 14 alpha-demethylase by RS-21607 in vitro and in vivo.

The discovery of selective lanosterol 14 alpha-demethylase inhibitors may lead to novel hypolipidemic drugs. RS-21607, (2S,4S)-cis-2[1H-imidazol-1-yl)methyl]-2-[2-(4-chlorophenyl)ethyl]-4- [[(4-aminophenyl)thio]methyl]-1,3-dioxolane, was characterized as a tight-binding, competitive inhibitor of lanosterol 14 alpha-demethylase purified from rat liver. The apparent Ki was determined to be 840 pM and found to be similar in hepatic microsomes from human, rat, and hamster. RS-21607, which contains two chiral centers, was a more effective lanosterol 14 alpha-demethylase inhibitor than its three stereoisomers. In vitro, RS-21607 had a greater affinity for lanosterol 14 alpha-demethylase than the other cytochromes P450 evaluated: CYP7, CYP27, CYP11A1, CYP19, CYP17, CYP11B1, CYP21, CYP3A4, CYP4A, CYP2D6, CYP1A2, CYP2C9, and 27-hydroxycholesterol 7 alpha-hydroxylase. The other stereoisomers were not as selective as RS-21607. Doses of 3-30 mg/kg RS-21607 given orally to hamsters caused a dose-dependent decrease in cholesterol biosynthesis with a corresponding accumulation of 24,25-dihydrolanosterol. RS-21607 inhibited the enzyme and cholesterol biosynthesis in hamster liver by 50% at 18 h following a 30 mg/kg oral dose. This was interpreted to indicate that RS-21607 is able to distribute to the site of action in hamsters and inhibit the target enzyme. In the same dose range, the plasma concentrations of testosterone, corticosterone, and progesterone, the endpoints for the cytochromes P450 involved in steroid biosynthesis, were relatively unaffected. These data show RS-21607 to be an effective and selective inhibitor of lanosterol 14 alpha-demethylase, both in vivo and in vitro. RS-21607 interacted with the purified enzyme to produce a type II binding spectrum, consistent with an interaction between the imidazole moiety and the heme. The electrostatic contribution of the imidazole binding was investigated using the desimidazole analog of RS-21607. The apparent Ki for the desimidazole compound (65 microM) was similar to the apparent Km for the substrate DHL (79 microM). Together, these data confirm that the ligand attached to the imidazole in RS-21607 is a good non-sterol substitute for DHL, i.e., binding to the enzyme with similar affinity, and that the coordination of the imidazole to the heme provides a major electrostatic contribution for the inhibition of lanosterol 14 alpha-demethylase by RS-21607. RS-21607 was also observed to increase the accumulation of 3 beta-hydroxy-24,25-dihydrolanost-8-en-32-al, the second intermediate in the multistep oxidation, but not the first intermediate. 24,25-dihydrolanost-8-ene-3 beta,32-diol.(ABSTRACT TRUNCATED AT 400 WORDS)

Accumulation of intermediates and isotopically sensitive enolization distinguish between aromatase (cytochrome P450 CYP19) from rat ovary and human placenta.

Aromatase activity in microsomes from human placenta (HPM) and rat ovary (ROM) was compared by measuring the accumulation of 19-oxygenated intermediates, the effect of tritium substitution upon rates, and the distribution of tritium in products. A considerable accumulation of intermediates (19-hydroxyandrogen and 19-al-androgen) and a lag in product formation (estrogen and water) was observed with ROM but not HPM. Addition of purified NADPH-cytochrome P450 reductase to ROM decreased the accumulation of 19-hydroxyandrostenedione and increased the rate of estrone formation. This difference could not be attributed to the microsomal reductase concentration since its concentration was similar in both tissues. Estrogen formation by aromatase from these tissues was not associated with a significant kinetic isotope effect when androstenedione was labeled with tritium at C-1 and C-2. Isotopically sensitive switching (branching) from the 19-al-androstenedione enzyme complex to form free 19-al-androstenedione rather than estrogen was not observed. These data suggest that for aromatase in both tissues, an enzymatic step exists between the 19-al-androstenedione intermediate and hydrogen abstraction or enolization that has a large commitment to catalysis. The distribution of tritium into the products, water and estrogen, was dependent upon substrate, enzyme source, and position of the label. Incubation of 1 beta, 2 beta-[3H]androstenedione and testosterone with ROM and 1 beta,2 beta-[3H]testosterone with HPM resulted in approximately 50% of the label being retained in the estrogen and 50% being lost in water. The majority of the label was lost in water upon incubation of 1 beta-labeled substrates with microsomes from both sources. Unexpectedly, no label was lost to water upon incubation of the specifically 1 alpha,2 alpha-labeled substrates with either enzyme source. Only incubation of 1 beta,2 beta-[3H]androstenedione with HPM resulted in loss of tritium from the 2-position. These data were interpreted to indicate that, for androstenedione metabolism by ROM and testosterone metabolism by both ROM and HPM, enolization occurs nonspecifically in an isotopically sensitive manner following deformylation, but for HPM metabolism of androstenedione enolization occurs in an enzyme-assisted manner. The studies show that aromatase located in ROM differs from that in HPM by its accumulation of intermediates and inability to selectively remove the 2 beta-tritium from androstenedione.

PAS IV, an integral membrane protein of mammary epithelial cells, is related to platelet and endothelial cell CD36 (GP IV).

PAS IV is a 78-kDa (bovine) to 80-kDa (human) integral membrane glycoprotein of unknown function which is found in mammary epithelial cells. We now report the purification of human PAS IV and native bovine PAS IV from the milk fat globule membrane (MFGM), a preparation of apical plasmalemma from epithelial cells of lactating mammary tissue. N-Terminal sequence analyses of human and bovine PAS IV revealed homology to the N-terminal sequence of the 88-kDa human endothelial and platelet glycoprotein CD36. The similarity of MFGM PAS IV to platelet CD36 was further established by immunoblots of purified platelet CD36 and MFGM PAS IV with MFGM PAS IV specific antiserum. The removal of N-linked oligosaccharides from PAS IV and CD36 by treatment with endoglycosidase F reduced the apparent Mr of both proteins to approximately 57,000. These data suggest that PAS IV and CD36 are similar if not identical polypeptides that undergo cell type specific glycosylation.