L-Tyrosine beta-naphthylamide is a potent competitive inhibitor of tyramine N-(hydroxycinnamoyl)transferase in vitro

L-Tyrosine beta-naphthylamide, a synthetic substrate designed to measure tyrosine aminopeptidase activity, is a potent inhibitor of hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)transferase (THT) purified from elicited tobacco cell-suspension cultures. The inhibition is competitive, with the inhibitor binding reversibly to the tyramine binding site of the enzyme. Similar results were obtained with THT extracted from elicited potato cell-suspension cultures. Ki values were found to be 0.66 microM for the enzyme from tobacco and 0.3 microM for the enzyme from potato. L-Tyrosine 7-amido-4-methylcoumarin, a fluorogenic substrate for tyrosine aminopeptidases, the structure of which is close to that of L-tyrosine beta-naphthylamide. was also a powerful inhibitor, but slightly less effective with Ki values of 0.72 and 0.42 microM for tobacco and potato THT, respectively. L-Tyrosine beta-naphthylamide was rapidly hydrolysed when fed in vivo to tobacco or potato cell cultures or when incubated in crude enzymic extracts prepared from these cultures. This hydrolysis, which is presumably catalysed by aminopeptidases, precludes the use of L-tyrosine amides as inhibitors of THT in vivo.     

An angiotensin converting enzyme inhibitor is a tight-binding slow substrate of carboxypeptidase A

Carboxypeptidase A-catalyzed hydrolysis of peptides and depsipeptides is competitively inhibited by N-(1-carboxy-5-t-butyloxycarbonylaminopentyl)-L-phenylalanine (Boc-CA-Phe, Ki = 1.3 microM) and the angiotensin converting enzyme inhibitor, N-(1-carboxy-5-carbobenzoxyaminopentyl)-glycyl-L-phenylalanine (Z-CA-Gly-Phe, Ki = 4.5 microM). The latter compound is actually a slow substrate of carboxypeptidase. Indirect observation of inhibitor binding by stopped-flow measurement of radiationless energy transfer between carboxypeptidase tryptophans and dansylated substrates reveals slow binding for both compounds. The visible absorption spectrum of the complex of cobalt(II)-substituted carboxypeptidase and Z-CA-Gly-Phe, which differs from the corresponding spectrum of the Boc-CA-Phe complex, is remarkable in its resemblance to the spectrum of the complex between Co(II)carboxypeptidase and a transient intermediate previously observed during hydrolysis of peptide substrates. The spectrum slowly changes to that of the free enzyme indicating hydrolysis. Chromatographic quantitation of substrate and products confirms that carboxypeptidase converts Z-CA-Gly-Phe to Z-CA-Gly and L-Phe with an apparent kcat of 0.02 s-1. Absorption spectroscopy indicates that the Z-CA-Gly-Phe-Co(II)carboxypeptidase spectrum is not that of bound products. Moreover, spectral titrations indicate that the products (both with spectral Ki values of about 3 mM), as well as D-Phe, compete for the same site on the enzyme.     

Enzymes of de novo pyrimidine biosynthesis in Babesia rodhaini

The pathway of de novo pyrimidine biosynthesis in the rodent parasitic protozoa Babesia rodhaini has been investigated. Specific activities of five of the six enzymes of the pathway were determined: aspartate transcarbamylase (ATCase: E.C. 2.1.3.2); dihydroorotase (DHOase: E.C. 3.5.2.3); dihydroorotate dehydrogenase (DHO-DHase: E.C. 1.3.3.1); orotate phosphoribosyltransferase (OPRTase: E.C. 2.4.2.10); and orotidine-5'-phosphate decarboxylase (ODCase: E.C. 4.1.1.23). Michaelis constants for ATCase, DHO-DHase, OPRTase, and ODCase were determined in whole homogenates. Several substrate analogs were also investigated as inhibitors and inhibitor constants determined. N-(phosphonacetyl)-L-aspartate was shown to be an inhibitor of the ATCase with an apparent Ki of 7 microM. Dihydro-5-azaorotate inhibited the DHO-DHase (Ki, 16 microM) and 5-azaorotate (Ki, 21 microM) was an inhibitor of the OPRTase. The UMP analog, 6-aza-UMP (Ki, 0.3 microM) was a potent inhibitor of ODCase, while lower levels of inhibition were found with the product, UMP (Ki, 120 microM) and the purine nucleotide, XMP (Ki, 95 microM). Additionally, menoctone, a ubiquinone analog, was shown to inhibit DHO-DHase.     

Purine nucleoside phosphorylase from Mycobacterium tuberculosis. Analysis of inhibition by a transition-state analogue and dissection by parts.

Purine salvage pathways are predicted to be present from the genome sequence of Mycobacterium tuberculosis. The M. tuberculosis deoD gene encodes a presumptive purine nucleoside phosphorylase (PNP). The gene was cloned, expressed, purified, and found to exhibit PNP activity. Purified M. tuberculosis PNP is trimeric, similar to mammalian PNP's but unlike the hexameric Escherichia coli enzyme. Immucillin-H is a rationally designed analogue of the transition state that has been shown to be a potent inhibitor of mammalian PNP's. This inhibitor also exhibits slow-onset inhibition of M. tuberculosis PNP with a rapid, reversible inhibitor binding (K(i) of 2.2 nM) followed by an overall dissociation constant (K(i)) of 28 pM, yielding a K(m)/K(i) value of 10(6). Time-dependent tight binding of the inhibitor occurs with a rate of 0.1 s(-)(1), while relaxation of the complex is slower at 1.4 x 10(-)(3) s(-)(1). The pH dependence of the K(i) value of immucillin-H to the M. tuberculosis PNP suggests that the inhibitor binds as the neutral, unprotonated form that is subsequently protonated to generate the tight-binding species. The M. tuberculosis enzyme demonstrates independent and equivalent binding of immucilin-H at each of the three catalytic sites, unlike mammalian PNP. Analysis of the components of immucillin-H confirms that the inhibition gains most of its binding energy from the 9-deazahypoxanthine group (K(is) of 0.39 microM) while the 1,4-dideoxy-1,4-iminoribitol binds weakly (K(is) of 2.9 mM). Double-inhibition studies demonstrate antagonistic binding of 9-deazahypoxanthine and iminoribitol (beta = 13). However, the covalent attachment of these two components in immucillin-H increases equilibrium binding affinity by a factor of >14 000 (28 pM vs 0.39 microM) compared to 9-deazahypoxanthine alone, and by a factor of >10(8) compared to iminoribitol alone (28 pM vs 2.9 mM), from initial velocity measurements. The structural basis for M. tuberculosis PNP inhibition by immucillin-H and by its component parts is reported in the following paper [Shi, W., Basso, L. A., Santos, D. S., Tyler, P. C., Furneaux, R. H., Blanchard, J. S., Almo, S. C., and Schramm, V. L. (2001) Biochemistry 40, 8204-8215].     

Direct photolabeling of the EcoRII methyltransferase with S-adenosyl-L-methionine

Ultraviolet irradiation of EcoRII methyltransferase in the presence of its substrate, S-adenosyl-L-methionine (AdoMet), results in the formation of a stable enzyme-substrate adduct. This adduct can be demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after irradiation of the enzyme in the presence of either [methyl-3H]AdoMet or [35S]AdoMet. The extent of photolabeling is low. Under optimal conditions, 4.5 pmol of [3H]AdoMet is incorporated into 100 pmol of enzyme. Use of the 8-azido derivative of AdoMet as the photolabeling substrate increases the incorporation by approximately 2-fold. However, this adduct, unlike the one formed with AdoMet, is not stable when treated with thiol reagents or precipitated with trichloroacetic acid. A catalytically active conformation of the enzyme is needed for AdoMet photolabeling. Heat-inactivated enzyme or proteins for which AdoMet is not a substrate or cofactor do not undergo adduct formation. Two other methyltransferases, MspI and dam methylases are also shown to form adducts with AdoMet upon UV irradiation. The binding constant of the EcoRII methyltransferase for AdoMet determined with the photolabeling reaction is 11 microM, which is similar to the binding constant of 9 microM previously reported (Friedman, S. (1986) Nucleic Acids Res. 14, 4543-4556). The AdoMet analogs S-adenosyl-L-homocysteine (Ki = 0.83 microM) and sinefungin (Ki = 4.3 microM) are effective inhibitors of photolabeling, whereas S-adenosyl-D-homocysteine (Ki = 46 microM) is a poor inhibitor. These experiments indicate that AdoMet becomes covalently bound at the AdoMet-binding site on the enzyme molecule. The EcoRII methyltransferase-AdoMet adduct is very stable and could be used to identify the AdoMet-binding site on DNA methyltransferases.     

Active site directed N-carboxymethyl peptide inhibitors of a soluble metalloendopeptidase from rat brain

A soluble metalloendopeptidase isolated from rat brain preferentially cleaves bonds in peptides having aromatic residues in the P1 and P2 position. An additional aromatic residue in the P3' position greatly increases the binding affinity of the substrate, suggesting the presence of an extended substrate recognition site in the enzyme, capable of binding a minimum of five amino acid residues [Orlowski, M., Michaud, C., & Chu, T.G. (1983) Eur. J. Biochem. 135, 81-88]. A series of N-carboxymethyl peptide derivatives structurally related to model substrates and containing a carboxylate group capable of coordinating with the active site zinc atom were synthesized and tested as potential inhibitors. One of these inhibitors, N-[1(RS)-carboxy-2-phenylethyl]-Ala-Ala-Phe-p-aminobenzoate, was found to be a potent competitive inhibitor of the enzyme with a Ki of 1.94 microM. The two diastereomers of this inhibitor were separated by high-pressure liquid chromatography. The more potent diastereomer had a Ki of 0.81 microM. The inhibitory potency of the less active diastereomer was lower by 1 order of magnitude. Decreasing the hydrophobicity of the residue binding the S1 subsite of the enzyme by, for example, replacement of the phenylethyl group with a methyl residue decreased the inhibitory potency by almost 2 orders of magnitude. Deletion of the carboxylate group decreased the inhibitory potency by more than 3 orders of magnitude. Shortening the inhibitor chain by a single alanine residue had a similar effect. Binding of the inhibitor to the enzyme increased its thermal stability.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization df a [3H]Glycine Recognition Site as a Modulatory Site of the N-Methyl-D-Aspartate Receptor Complex

A [3H]glycine recognition site in rat brain synaptic plasma membranes (SPM) has been identified, having characteristics expected of a modulatory component of the N-methyl-D-aspartate receptor complex. Incubation of SPM with [3H]glycine for 10 min at 2 degrees C results in saturable, reversible binding with a KD of 0.234 microM and a Bmax of 9.18 pmol/mg. A pharmacological analysis of this binding site indicates that D-serine (Ki = 0.27 microM), D-alanine (Ki = 1.02 microM), and D-cycloserine (Ki = 2.33 microM) are potent inhibitors of binding, whereas the corresponding L isomers have significantly less activity (Ki = 25.4 microM, 15.9 microM, and greater than 100 microM, respectively). Inactive at concentrations of up to 100 microM were strychnine, L-valine, N,N-dimethylglycine, aminomethylphosphonate, and aminomethylsulfonate. The active compounds were analyzed further for their ability to stimulate [3H]1-[1-(2-thienyl)cyclohexyl]piperidine [( 3H]TCP) binding to Triton X-100-washed SPM. Results indicate that the affinity of the compounds for the [3H]glycine recognition site correlates with the ability of these analogues to stimulate [3H]TCP binding.     

Exosite-mediated substrate recognition of factor IX by factor XIa. The factor XIa heavy chain is required for initial recognition of factor IX

Studies of the mechanisms of blood coagulation zymogen activation demonstrate that exosites (sites on the activating complex distinct from the protease active site) play key roles in macromolecular substrate recognition. We investigated the importance of exosite interactions in recognition of factor IX by the protease factor XIa. Factor XIa cleavage of the tripeptide substrate S2366 was inhibited by the active site inhibitors p-aminobenzamidine (Ki 28 +/- 2 microM) and aprotinin (Ki 1.13 +/- 0.07 microM) in a classical competitive manner, indicating that substrate and inhibitor binding to the active site was mutually exclusive. In contrast, inhibition of factor XIa cleavage of S2366 by factor IX (Ki 224 +/- 32 nM) was characterized by hyperbolic mixed-type inhibition, indicating that factor IX binds to free and S2366-bound factor XIa at exosites. Consistent with this premise, inhibition of factor XIa activation of factor IX by aprotinin (Ki 0.89 +/- 0.52 microM) was non-competitive, whereas inhibition by active site-inhibited factor IXa beta was competitive (Ki 0.33 +/- 0.05 microM). S2366 cleavage by isolated factor XIa catalytic domain was competitively inhibited by p-aminobenzamidine (Ki 38 +/- 14 microM) but was not inhibited by factor IX, consistent with loss of factor IX-binding exosites on the non-catalytic factor XI heavy chain. The results support a model in which factor IX binds initially to exosites on the factor XIa heavy chain, followed by interaction at the active site with subsequent bond cleavage, and support a growing body of evidence that exosite interactions are critical determinants of substrate affinity and specificity in blood coagulation reactions.     

Inhibitory Effects of the Monoamine Oxidase Inhibitor Tranylcypromine on the Cytochrome P450 Enzymes CYP2C19, CYP2C9, and CYP2D6

1. The inhibitory effects of tranylcypromine, a nonselective irreversible inhibitor of monoamine oxidase (MAO), on three cytochrome P450 (CYP) enzymes, namely CYP2C9, CYP2C19, and CYP2D6, have been evaluated in vitro. 2. The studies were conducted using cDNA-expressed human CYP enzymes and probe substrates. 3. A range of substrate concentrations was coincubated with a range of tranylcypromine concentrations in the presence of each of the CYP enzymes at 37 degrees C for a predetermined period of time. Product concentrations were quantified by HPLC with UV detection. 4. The results demonstrated that tranylcypromine is a competitive inhibitor of CYP2C19 (Ki = 32 microM) and CYP2D6 (Ki = 367 microM) and a noncompetitive inhibitor of CYP2C9 (Ki = 56 microM). 5. None of these inhibitory effects are considered clinically significant at usual therapeutic doses. However, in certain situations such as high dose tranylcypromine therapy, or in poor metabolizers of CYP2C19 substrates, clinically significant interactions might occur, particularly when tranylcypromine is coadministered with drugs with a narrow therapeutic index.     

Inhibition of urease by cyclic beta-triketones and fluoride ions

Competitive inhibition of soybean urease by 11 cyclic beta-triketones was studied in aqueous solutions at pH 7.4 and 36 degrees C. This process was characterized quantitatively by the inhibition constant (Ki), which showed a strong dependence on the structure of organic chelating agents (nickel atoms in urease) and varied from 58.4 to 847 microM. Under similar conditions, the substrate analogue (hydroxyurea) acted as a weak urease inhibitor (Ki = 6.47 mM). At 20 degrees C, competitive inhibition of urease with the ligand of nickel atoms (fluoride anion) was pH-dependent. At pH 3.85-6.45, the value of Ki for the process ranged from 36.5 to 4060 microM. Three nontoxic cyclic beta-triketones with Ki values of 58.4, 71.4, and 88.0 microM (36 degrees C) were the most potent inhibitors of urease. Their efficacy was determined by the presence of three >C=O- groups in the molecule and minimum steric hindrances to binding with metal sites in soybean urease.     

Interaction of t-Butylbicyclophosphorothionate with γ-Aminobutyric Acid-Gated Chloride Channels in Cultured Cerebral Neurons

The role of t-butylbicyclophosphorothionate (TBPS) as an antagonist of gamma-aminobutyric acid (GABA) was studied with primary cultures of neurons from the chick embryo cerebrum. The addition of GABA stimulated the uptake of 36Cl- by neurons and the dose dependence of this effect followed hyperbolic kinetics with a K0.5 = 1.3 microM for GABA. TBPS proved to be a potent inhibitor of GABA-dependent Cl- uptake (IC50 = 0.30 microM). Analysis of the kinetics of this process revealed that TBPS is a noncompetitive inhibitor (Ki = 0.15 microM) with respect to GABA. Scatchard analysis of direct binding of [35S]TBPS to membranes isolated from neuronal cultures gave curvilinear plots. These could be resolved by nonlinear regression methods into two components with KD values of 3.1 nM and 270 nM. The TBPS binding constant for this lower affinity site agreed well with the IC50 and Ki values for inhibition of Cl- flux, suggesting that this site is physiologically relevant to GABA antagonism. GABA was a noncompetitive displacer of [35S]TBPS binding to the lower affinity site. The Ki value for this displacement by GABA (1.7 microM) was comparable to the value for GABA enhancement of Cl- flux. The binding of [35S]TBPS to its low-affinity site on neuronal membranes was ninefold higher in the presence of Cl- than with gluconate, an impermeant anion. The rank order for anion stimulation of [35S]TBPS binding was Br- greater than or equal to SCN- greater than Cl- greater than or equal to NO3- greater than I- greater than F- greater than gluconate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Unsaturated long-chain N-acyl-vanillyl-amides (N-AVAMs): vanilloid receptor ligands that inhibit anandamide-facilitated transport and bind to CB1 cannabinoid receptors.

We investigated the effect of changing the length and degree of unsaturation of the fatty acyl chain of N-(3-methoxy-4-hydroxy)-benzyl-cis-9-octadecenoamide (olvanil), a ligand of vanilloid receptors, on its capability to: (i) inhibit anandamide-facilitated transport into cells and enzymatic hydrolysis, (ii) bind to CB1 and CB2 cannabinoid receptors, and (iii) activate the VR1 vanilloid receptor. Potent inhibition of [(14)C]anandamide accumulation into cells was achieved with C20:4 n-6, C18:3 n-6 and n-3, and C18:2 n-6 N-acyl-vanillyl-amides (N-AVAMs). The saturated analogues and Delta(9)-trans-olvanil were inactive. Activity in CB1 binding assays increased when increasing the number of cis-double bonds in a n-6 fatty acyl chain and, in saturated N-AVAMs, was not greatly sensitive to decreasing the chain length. The C20:4 n-6 analogue (arvanil) was a potent inhibitor of anandamide accumulation (IC(50) = 3.6 microM) and was 4-fold more potent than anandamide on CB1 receptors (Ki = 0.25-0.52 microM), whereas the C18:3 n-3 N-AVAM was more selective than arvanil for the uptake (IC(50) = 8.0 microM) vs CB1 receptors (Ki = 3.4 microM). None of the compounds efficiently inhibited [(14)C]anandamide hydrolysis or bound to CB2 receptors. All N-AVAMs activated the cation currents coupled to VR1 receptors overexpressed in Xenopus oocytes. In a simple, intact cell model of both vanilloid- and anandamide-like activity, i.e., the inhibition of human breast cancer cell (HBCC) proliferation, arvanil was shown to behave as a "hybrid" activator of cannabinoid and vanilloid receptors.     

Asymmetry of the red cell anion exchange system. Different mechanisms of reversible inhibition by N-(4-azido-2-nitrophenyl)-2- aminoethylsulfonate (NAP-taurine) at the inside and outside of the membrane

In the dark, the photoaffinity reagent, N-(4-azido-2-nitrophenyl)-2- aminoethylsulfonate (NAP-taurine), acts as a reversible inhibitor of red cell anion exchange when it is present either within the cell or in the external solution. A detailed analysis of the inhibition kinetics, however, reveals substantial differences in the responses to the probe at the two sides of the membrane. On the inside of the cell, NAP- taurine is a relatively low affinity inhibitor of chloride exchange (Ki = 370 microM). Both the effects of chloride on NAP-taurine inhibition and the affinity of NAP-taurine for the system as a substrate are consistent with the concept that internal NAP-taurine competes with chloride for the substrate site of the anion exchange system. External NAP-taurine, on the other hand, is a far more potent inhibitor of chloride exchange (Ki = 20 microM). It acts at a site of considerably lower affinity for chloride than the substrate site, probably the modifier site, at which halide anions are reported to cause a noncompetitive inhibition of chloride transport. NAP-taurine therefore seems to interact preferentially with either the substrate or modifier site of the transport system, depending on the side of the membrane at which it is present. It is suggested that the modifier site is accessible to NAP-taurine only from the outside whereas the transport site may be accessible from either side.     

Histochemical modification of the active site of succinate dehydrogenase with N-acetylimidazole

The kinetics of acetylation of mitochondrial succinate dehydrogenase [EC 1.3.99.1] in the two fibre types (A and C) of rat gastrocnemius with N-acetylimidazole was studied by a newly modified histochemical technique. Acetylimidazole partially inactivated the enzyme, but subsequent deacetylation with hydroxylamine restored the enzyme activity completely. Inactivation of the enzyme by acetylimidazole was prevented by malonate, which is a competitive inhibitor of the enzyme. The value of the inhibition constant (Ki = 34 microM) for malonate, obtained from the dependence of the pseudo-first order rate constant of acetylation of the enzyme with acetylimidazole on the malonate concentration, was in good agreement with the Ki value (33 microM) obtained by a different method, the dependence of the initial velocity of succinate oxidation by the dehydrogenase on the substrate concentration in the presence of malonate. These findings suggest that a tyrosyl residue is located in the malonate binding site (the active site) of succinate dehydrogenase in the gastrocnemius and plays a role in substrate binding, but is not a catalytic group.     

Active site specificity of the adenylate cyclase catalytic unit from bovine caudate nucleus

ATP analogues were used to study the active site specificity of the catalytic unit (C) of solubilized and partially purified bovine brain caudate nucleus adenylate cyclase. Phenylenediamine ATP (PD-ATP), 8-azido ATP (8-N3ATP), chromium(III) 3'-beta-alanylarylazido ATP (CrATPa), and 2',3'-dialdehyde ATP (oATP) are competitive inhibitors of C in the presence of the substrate MnATP and the activator forskolin. (Km for MnATP is 50 +/- 11 microM, n = 13). The Ki values determined under initial velocity conditions are: PD-ATP, Ki = 695 +/- 60 microM, n = 5; 8-N3ATP, Ki = 155 +/- 23 microM, n = 5; CrATPa, Ki = 7 +/- 3 microM, n = 2; oATP, Ki = 42 +/- 5 microM, n = 3. Irradiation of 100 microM 8-N3ATP by UV light (254 nm) causes the first-order loss of reagent either in the presence or absence of C. Concomitant irreversible inhibition of C in the presence of 8-N3ATP was more complex and asymptotically approached 50% within 4-6 min. Loss of C activity in controls was 10-20%. The fraction of C covalently modified by 8-N3ATP, alpha, was calculated for each time point of irradiation for an increasing initial concentration ([A]o) of 8-N3ATP. Extrapolated to infinite time of photolysis, the value of alpha reached a final level, termed alpha t whose magnitude depended on [A]o. From these data we calculated an apparent KD of 4.5 microM for 8-N3ATP. ATP protected against the irreversible inhibition due to 8-N3ATP. These data are most consistent with a mechanism of photoaffinity labeling involving equilibrium binding and covalent insertion of 8-N3ATP into the active site. These results indicate that the active site binds analogues of ATP which are considerably modified in the adenine, ribose, and gamma-phosphate portions and that the affinity of C for these analogues is within an order of magnitude of the Km for ATP.     

Atomic dissection of the hydrogen bond network for transition-state analogue binding to purine nucleoside phosphorylase

Immucillin-H (ImmH) and immucillin-G (ImmG) were previously reported as transition-state analogues for bovine purine nucleoside phosphorylase (PNP) and are the most powerful inhibitors reported for the enzyme (K(i) = 23 and 30 pM). Sixteen new immucillins are used to probe the atomic interactions that cause tight binding for bovine PNP. Eight analogues of ImmH are identified with equilibrium dissociation constants of 1 nM or below. A novel crystal structure of bovine PNP-ImmG-PO(4) is described. Crystal structures of ImmH and ImmG bound to bovine PNP indicate that nearly every H-bond donor/acceptor site on the inhibitor is fully engaged in favorable H-bond partners. Chemical modification of the immucillins is used to quantitate the energetics for each contact at the catalytic site. Conversion of the 6-carbonyl oxygen to a 6-amino group (ImmH to ImmA) increases the dissociation constant from 23 pM to 2.6 million pM. Conversion of the 4'-imino group to a 4'-oxygen (ImmH to 9-deazainosine) increases the dissociation constant from 23 pM to 2.0 million pM. Substituents that induce small pK(a) changes at N-7 demonstrate modest loss of affinity. Thus, 8-F or 8-CH(3)-substitutions decrease affinity less than 10-fold. But a change in the deazapurine ring to convert N-7 from a H-bond donor to a H-bond acceptor (ImmH to 4-aza-3-deaza-ImmH) decreases affinity by >10(7). Introduction of a methylene bridge between 9-deazahypoxanthine and the iminoribitol (9-(1'-CH(2))-ImmH) increased the distance between leaving and oxacarbenium groups and increased K(i) to 91 000 pM. Catalytic site energetics for 20 substitutions in the transition-state analogue are analyzed in this approach. Disruption of the H-bond pattern that defines the transition-state ensemble leads to a large decrease in binding affinity. Changes in a single H-bond contact site cause up to 10.1 kcal/mol loss of binding energy, requiring a cooperative H-bond pattern in binding the transition-state analogues. Groups involved in leaving group activation and ribooxacarbenium ion stabilization are central to the H-bond network that provides transition-state stabilization and tight binding of the immucillins.     

Substrate and inhibitor specificity of 3-hydroxy-3-methylglutaryl-CoA reductase determined with substrate-analogue CoA-thioesters and CoA-thioethers.

1) Analogues of 3-hydroxy-3-methylglutaryl-CoA were prepared in which the substituents at C-3 of the acyl residue were altered. The same analogues were additionally modified by replacement of the thioester oxygen by hydrogen to yield reduction-resistant CoA-thioethers. The interaction of both types of CoA derivatives with a 58-kDa catalytic fragment of human 3-hydroxy-3-methylglutaryl-CoA reductase was studied. 2) This enzyme reduces glutaryl-CoA at a very low rate whereas 3-hydroxyglutaryl-CoA is well reduced, the maximal rate of reduction being 7% that of the physiological substrate. Only half of total 3-hydroxyglutaryl-CoA was attacked, thus reflecting the stereo-specificity of the enzyme for (3S)-3-hydroxy-3-methylglutaryl-CoA. The results invalidate the hitherto assumed absolute substrate specificity of the enzyme. 3) The affinity of both 3-hydroxyglutaryl-CoA and its thioether variant S-(4-carboxy-3-hydroxybutyl)CoA to the reductase, Ki = 0.3 microM and Ki = 0.4 microM, respectively, is higher than that of the physiological substrate, Km = 1.5 microM (data related to (S)-diastereomer). The results show for the first time that the methyl-group effect observed with the inhibitor lovastatin is an intrinsic property of the enzyme. 4) All of the prepared CoA derivatives are purely competitive inhibitors of the reductase, the affinities varying within a range of two powers of ten (Ki = 0.3-32 microM). On variation of the substituents at C-3 of the acyl residue of the physiological substrate the affinity of both CoA-thioesters and CoA-thioethers increases in the sequence CH2, C(CH3)2, CH(CH3), C(OH)CH3, CH(OH).     

Leukotriene C4 binding to rat lung membranes

A high affinity binding site for leukotriene C4 (LTC4), one component of slow reacting substance of anaphylaxis, has been identified in a membrane preparation from rat lung. As measured by a filtration technique, [3H]LTC4 binding was saturable, specific, reversible, and heat-sensitive. In the presence of 20 mM CaCl2, the dissociation constant (KD) was 41 +/- 9 nM and the maximum number of binding sites (Bmax) was 31 +/- 10 pmol/mg of protein. Specificity was demonstrated by competition studies in which LTC4 had a Ki of 40 nM against specifically bound [3H]LTC4, whereas leukotriene D4 (LTD4) had a Ki of 4 microM. The stereoisomers (5R, 6R) LTC4, (5S, 6S) LTC4, and (5R, 6S) LTC4 had Ki values 3-, 15-, and 25-fold higher than that of natural (5S, 6R) LTC4. Leukotrienes E4 and B4, several prostaglandins and fatty acids, glutathione, and platelet activating factor were even less effective with Ki values above 10 microM. A slow reacting substance of anaphylaxis antagonist, FPL 55712, which, in some systems, distinguishes LTC4- from LTD4-induced contractions, was a weak competitor with a Ki of 16 microM. Serine-borate complex which inhibits gamma-glutamyl transpeptidase, an enzyme responsible for LTC4 metabolism, did not alter binding. In addition, 100 microM FPL 55712 did not reduce metabolism. These observations suggest that the binding observed for LTC4 may represent association with a physiological receptor for this molecule which has a relatively low affinity for LTD4.     

6-(N-benzoylamino)purine as a novel and potent inhibitor of xanthine oxidase: inhibition mechanism and molecular modeling studies

The inhibition of xanthine oxidase (XO) activity by the purine analogue 6-(N-benzoylamino)purine was evaluated and compared with the standard inhibitor, allopurinol and the parent compound adenine. 6-(N-benzoylamino)purine is a highly potent inhibitor of XO (IC50 = 0.45 microM) and comparable to allopurinol (IC50 = 0.80 microM). Furthermore, 6-(N-benzoylamino)purine neither produced any enzymatic superoxide nor reduced XO by an electron transfer reaction unlike allopurinol. 6-(N-benzoylamino)purine (Ki = 0.0475 microM) is about 10000-fold more potent as a XO inhibitor compared to the only known purine analogue 8-bromoxanthine (Ki = 400 microM). 6-(N-Benzoylamino)purine is a competitive inhibitor of XO and the inhibition was not completely reversed even at 100 microM xanthine concentration. The calculated interaction energy [Ecomplex - (Eligand + Eprotein)] of -30.5, -22.6, and -17.2 kcal/mol, respectively, of 6-(N-benzoylamino)purine, 8-bromoxanthine and the parent compound adenine provided the rationale for the better enzyme inhibitory activity of 6-(N-benzoylamino)purine. To understand the role of the benzamido group in the inhibition process, molecular docking studies were carried out and it was revealed that the hydrogen bonding interactions involving N-7 of the purine ring and the N-H of Arg880, N-H of the purine ring and OH of Thr1010, as well as non-bonded interactions of the benzamido group of 6-(N-benzoylamino)purine with amino acid residues Gly799, Glu802, Phe914, Ala1078, Ala1079 and Glu1261 in the active site of XO play an important role in the stabilization of the E-I complex.     

Antiplatelet effects of conjugated linoleic acid isomers

Conjugated diene isomers of linoleic acid (CLA) are normal constituents of certain foods and exhibit anticarcinogenic and antiatherogenic properties. In the present study, the effects of several CLA isomers on human platelet aggregation and arachidonic acid metabolism were examined. It was found that 9c,11t-CLA, 10t, 12c-CLA and 13-hydroxy-9c,11t-octadecadienoic acid (13-HODE) inhibited arachidonic acid- and collagen-induced platelet aggregation with I50s in the 5-7 microM range. The nonconjugated 9c, 12c-LA was about 300% and 50%, respectively, less potent an inhibitor with these aggregating agents. Using either thrombin or the calcium ionophore A23187 as aggregating agents, a CLA isomer mix was also found to be more inhibitory than 9c,12c-LA. The 9c,11t- and 10t,12c-CLA isomers as well as the CLA isomer mix inhibited formation of the proaggregatory cyclooxygenase-catalyzed product TXA2, as measured by decreased production of its inactive metabolite [14C]TXB2 from exogenously added [14C]arachidonic acid (I50s=9-16 microM). None of the CLA isomers tested inhibited production of the platelet lipoxygenase metabolite [14C]12-HETE. The additional presence of a hydroxyl group gave opposite results: 13-HODE (I50=3 microM) was about 4-fold more potent a cyclooxygenase inhibitor than the 9c,11t-CLA isomer but 9-HODE was 2- to 3-fold less effective an inhibitor (I50=34 microM) of [14C]TXB2 formation than the corresponding 10t,12c-CLA. In both the aggregation and arachidonic acid metabolism experiments, the inhibitory effects of CLA on platelets were reversible and dependent on the time of addition of either the aggregating agent or the [14C]arachidonic acid substrate. These studies suggest that CLA isomers may also possess antithrombotic properties.