Structural analysis of NSAID binding by prostaglandin H2 synthase: time-dependent and time-independent inhibitors elicit identical enzyme conformations

Nonsteroidal antiinflammatory drugs (NSAIDs) block prostanoid biosynthesis by inhibiting prostaglandin H(2) synthase (EC 1.14.99.1). NSAIDs are either rapidly reversible competitive inhibitors or slow tight-binding inhibitors of this enzyme. These different modes of inhibition correlate with clinically important differences in isoform selectivity. Hypotheses have been advanced to explain the different inhibition kinetics, but no structural data have been available to test them. We present here crystal structures of prostaglandin H(2) synthase-1 in complex with the inhibitors ibuprofen, methyl flurbiprofen, flurbiprofen, and alclofenac at resolutions ranging from 2.6 to 2.75 A. These structures allow direct comparison of enzyme complexes with reversible competitive inhibitors (ibuprofen and methyl flurbiprofen) and slow tight-binding inhibitors (alclofenac and flurbiprofen). The four inhibitors bind to the same site and adopt similar conformations. In all four complexes, the enzyme structure is essentially unchanged, exhibiting only minimal differences in the inhibitor binding site. These results argue strongly against hypotheses that explain the difference between slow tight-binding and fast reversible competitive inhibition by invoking global conformational differences or different inhibitor binding sites. Instead, they suggest that the different apparent modes of NSAID binding may result from differences in the speed and efficiency with which inhibitors can perturb the hydrogen bonding network around Arg-120 and Tyr-355.     

The ribonuclease inhibitors from porcine thyroid and liver are slow, tight-binding inhibitors of bovine pancreatic ribonuclease A

Ribonuclease inhibitors were purified from the latent ribonuclease fractions of porcine thyroid and liver and used to test the hypothesis that their inhibition of bovine pancreatic ribonuclease A is correctly described by tight-binding rather than Michaelis-Menton kinetics. Both proteins were found to act as slow, tight-binding inhibitors of the enzyme. These steady-state velocities also showed that both the thyroid and liver inhibitors were competitive inhibitors of bovine pancreatic ribonuclease A with Ki's of 0.1 and 0.4 nM, respectively. In contrast to interpretations based on Michaelis-Menton assumptions that show non-competitive inhibition, these results suggest that an enzyme:inhibitor:substrate complex does not exist.     

A novel approach to serine protease inhibition: kinetic characterization of inhibitors whose potencies and selectivities are dramatically enhanced by Zinc(II)

Serine proteases play a role in a variety of disease states and thus are attractive targets for therapeutic intervention. We report the kinetic characterization of a class of serine protease inhibitors whose potencies and selectivities are dramatically enhanced in the presence of Zn(II). The structural basis for Zn(II)-mediated inhibition of trypsin-like proteases has recently been reported [Katz, B. A., Clark, J. M., Finer-Moore, J. S., Jenkins, T. E., Johnson, C. R., Ross, M. J., Luong, C., Moore, W. R., and Stroud, R. M. (1998) Nature 391, 608-612]. A case study of the kinetic behavior of human tryptase inhibitors is provided to illustrate the general phenomenon of Zn(II)-mediated inhibition. Tryptase, Zn(II), and the inhibitor form a ternary complex which exhibits classic tight-binding inhibition. The half-life for release of inhibitor from the tryptase-Zn(II)-inhibitor complex has been measured for a number of inhibitors. Consistent with tight-binding behavior, potent tryptase inhibitors are characterized by extremely slow rates of dissociation from the ternary complex with half-lives on the order of hours. A model of human serum, designed to reproduce physiological levels of Zn(II), has been employed to evaluate the performance of Zn(II)-potentiated tryptase inhibitors under physiological conditions. We demonstrate that Zn(II)-mediated inhibition can be achieved at physiological Zn(II) levels.     

氟比洛芬酯联合地佐辛用于甲状腺手术术后镇痛的研究

摘要目的：研究氟比洛芬酯与地佐辛联合应用对于甲状腺手术患者术后的镇痛效果。方法：选择90例ASAI或II级甲状腺手术的患者（包括结节性甲状腺肿,甲状腺功能亢进,甲状腺癌）。年龄21．59岁,一般状态良好,随机分为三组：（FD组）氟比洛芬酯＋地佐辛组,（F组）氟比洛芬酯组,（D组）地佐辛组,每组30例。各组于手术结束前30rain分别缓慢静脉输注药物,FD组：氟比洛芬酯50mg＋地佐辛5mg;F组：氟比洛芬酯50mg;D组：地佐辛5mg。然后各组分别泵入不同的药物,FD组：氟比洛芬酯200mg＋地佐辛30mg,F组：氟比洛芬酯200mg,D组：地佐辛30mg,均以生理盐水稀释至100mL,以2ml／h的速率持续泵注48小时。分别记录各组各时间点：（T1）术后2h、（T2）术后6h、（T3）术后12h、（T4）术后24h、（T5）术后48h的心率（HR）、血氧饱和度（SPO：）及各时间点的疼痛评分（VAS）、镇静评分（Ramsay）,并记录出现的恶心、呕吐、眩晕等并发症的例数。结果：FD组、F组血流动力学较之D组平稳,D组出现一过性sP02下降。术后VAS评分D组明显高于FD组和F组（P〈0．05）,Ramsay评分FD组、F组低于D组（P〈0．05）。结论：在甲状腺手术术后镇痛的研究中,氟比洛芬酯与地佐辛联合应用,并发症少,镇痛良好,VAS、Ramsay等各项评分优于单纯应用氟比洛芬酯与地佐辛组。     

Kinetic determination of tight-binding impurities in enzyme inhibitors

A novel rate equation to characterize the dose-response behavior of a moderately potent ("classical") enzyme inhibitor contaminated with a very potent ("tight-binding") impurity is derived. Mathematical properties of the new rate equation show that, for such contaminated materials, experimentally observed I(50) values are ambiguous. The four-parameter logistic equation, conventionally used to determine I(50) values, cannot be used to detect the presence of tight-binding impurities in inhibitor samples. In contrast, fitting the newly derived rate equation to inhibitor dose- response curves can, in favorable cases, reveal whether the unknown material is chemically homogeneous or whether it is contaminated with a tight-binding impurity. The limitations of our method with respect to the detectable range of inhibition constants (both classical and tight-binding) were examined by using Monte-Carlo simulations. To test the new analytical procedure experimentally, we added a small amount (0.02 mole%) of a tight-binding impurity (K(i)=0.065 nM) to an otherwise weak inhibitor of human mast-cell tryptase (K(i)=50.4 microM). The resulting material was treated as "unknown." Our kinetic equation predicts that such adulterated material should show I(50)=0.40 microM, which was identical to the experimentally observed value. The best-fit value of the apparent inhibition constants for the tight-binding inhibitor was K(i)=(0.107+/-0.035)nM, close to the true value of 0.065 nM.     

Directed coevolution of stability and catalytic activity in calcium-free subtilisin

We have coevolved high activity and hyperstability in subtilisin by sequentially randomizing 12 amino acid positions in calcium-free subtilisin. The optimal amino acid for each randomized site was chosen based on stability and catalytic properties and became the parent clone for the next round of mutagenesis. Together, the 12 selected mutations increased the half-life of calcium-free subtilisin at elevated temperature by 15,000-fold. The catalytic properties of the mutants were examined against a range of substrates. In general, only mutations occurring at or near the substrate-binding surface have measurable effects on catalytic constants. No direct influence of stability on catalytic properties was observed. A high-stability mutant, Sbt140, was a more efficient enzyme in terms of k(cat)/K(m) than a commercial version of subtilisin across a range of substrates but had a lower k(cat) against tight-binding substrates. The reason for this behavior was discerned by examining microscopic rate constants for the hydrolysis of a tight-binding peptide substrate. Burst kinetics were observed for this substrate, indicating that acylation is not rate-limiting. Although acylation occurs at the rate of substrate binding, k(cat) is attenuated by the slow release of the N-terminal product. Natural evolution appears to have optimized catalytic activity against a range of sequences by achieving a balance between substrate binding and the rate of release of the N-terminal product.     

NO-flurbiprofen reduces amyloid-β, is neuroprotective in cell culture, and enhances cognition in response to cholinergic blockade

The non-steroidal anti-inflammatory drug flurbiprofen is a selective amyloid lowering agent which has been studied clinically in Alzheimer's disease. HCT-1026 is an ester prodrug of flurbiprofen incorporating a nitrate carrier moiety that in vivo provides NO bioactivity and an improved safety profile. In vitro, HCT-1026 retained the cyclooxygenase inhibitory and non-steroidal anti-inflammatory drug activity of flurbiprofen, but at concentrations at which levels of amyloid-β 1–42 amino acid were lowered by flurbiprofen, amyloid-β 1–42 amino acid levels were elevated 200% by HCT-1026. Conversely, at lower concentrations, HCT-1026 behaved as a selective amyloid lowering agent with greater potency than flurbiprofen. The difference in concentration–responses between flurbiprofen and HCT-1026 in vitro suggests different cellular targets; and in no case did a combination of nitrate drug with flurbiprofen provide similar actions. In vivo, HCT-1026 was observed to reverse cognitive deficits induced by scopolamine in two behavioral assays; activity that was also shown by a classical nitrate drug, but not by flurbiprofen. The ability to restore aversive memory and spatial working and reference memory after cholinergic blockade has been demonstrated by other agents that stimulate NO/cGMP signaling. These observations add positively to the preclinical profile of HCT-1026 and NO chimeras in Alzheimer's disease.     

The influence of brain inflammation upon neuronal adenosine A2B receptors

Alzheimer's disease (AD) is associated with glial activation and increased levels of pro-inflammatory cytokines. Epidemiological results suggest that anti-inflammatory therapies can slow the onset of AD. Adenosine, acting at type-2 receptors, is an effective endogenous anti-inflammatory agent that can modulate inflammation both in the periphery and the brain. We investigated changes in the expression of adenosine type-2B (A2B) receptors and a related intracellular second messenger during chronic brain inflammation and following treatment with the non-steroidal anti-inflammatory drug flurbiprofen and its nitric oxide (NO)-donating derivative, HCT1026. Chronic infusion of lipopolysaccharide (LPS) into the 4th ventricle of young rats induced brain inflammation that was associated with microglial activation and reduced neuronal immunoreactivity for adenosine A2B receptors in the cortex. Daily administration of HCT1026, but not flurbiprofen, reduced microglial activation, prevented the down-regulation of A2B receptors and elevated tissue levels of cAMP. The results suggest that a therapy using an NO-releasing NSAID might significantly attenuate the processes that drive the pathology associated with AD and that this process may involve the activation of adenosine A2B receptors.     

Effector-mediated alteration of substrate orientation in cytochrome P450 2C9

Cytochrome P450 2C9 (CYP2C9)-mediated flurbiprofen 4'-hydroxylation is activated by the presence of dapsone resulting in reduction of the K(m) for flurbiprofen hydroxylation and an increase in V(m). Previous spectral binding studies have demonstrated that the binding of flurbiprofen with CYP2C9 is increased (decrease in K(S)) by the presence of dapsone. We hypothesized that the two compounds are simultaneously in the active site with the presence of dapsone causing flurbiprofen to be oriented more closely to the heme. T(1) relaxation rates determined by NMR were used to estimate the distances of protons on these compounds from the paramagnetic heme-iron center. Samples contained 0.014 microM CYP2C9 and 145 microM flurbiprofen in the presence and absence of 100 microM dapsone. Estimated distances of various flurbiprofen protons from the heme ranged from 4.2 to 4.5 A in the absence of dapsone and from 3.2 to 3.8 A in the presence of dapsone. The 4' proton of flurbiprofen, the site of metabolism, showed one of the greatest differences in distance from the heme in the presence of dapsone, 3.50 A, as compared to the absence of dapsone, 4.41 A. Dapsone protons were less affected, being 4.40 A from the heme in the absence of flurbiprofen and 4.00-4.01 A from the heme in the presence of flurbiprofen. Molecular modeling studies were also performed to corroborate the relative orientations of flurbiprofen and dapsone in the active site of CYP2C9. Shift of the 4' proton of flurbiprofen closer to the heme iron of CYP2C9 in the presence of dapsone may play a role in activation.     

Inhibition of lipid A biosynthesis as the primary mechanism of CHIR-090 antibiotic activity in Escherichia coli

The deacetylation of UDP-3-O-[(R)-3-hydroxymyristoyl]-N-acetylglucosamine (UDP-3-O-acyl-GlcNAc) by LpxC is the committed reaction of lipid A biosynthesis. CHIR-090, a novel N-aroyl-l-threonine hydroxamic acid, is a potent, slow, tight-binding inhibitor of the LpxC deacetylase from the hyperthermophile Aquifex aeolicus, and it has excellent antibiotic activity against Pseudomonas aeruginosa and Escherichia coli, as judged by disk diffusion assays. We now report that CHIR-090 is also a two-step slow, tight-binding inhibitor of E. coli LpxC with Ki = 4.0 nM, Ki* = 0.5 nM, k5 = 1.9 min-1, and k6 = 0.18 min-1. CHIR-090 at low nanomolar levels inhibits LpxC orthologues from diverse Gram-negative pathogens, including P. aeruginosa, Neisseria meningitidis, and Helicobacter pylori. In contrast, CHIR-090 is a relatively weak competitive and conventional inhibitor (lacking slow, tight-binding kinetics) of LpxC from Rhizobium leguminosarum (Ki = 340 nM), a Gram-negative plant endosymbiont that is resistant to this compound. The KM (4.8 microM) and the kcat (1.7 s-1) of R. leguminosarum LpxC with UDP-3-O-[(R)-3-hydroxymyristoyl]-N-acetylglucosamine as the substrate are similar to values reported for E. coli LpxC. R. leguminosarum LpxC therefore provides a useful control for validating LpxC as the primary target of CHIR-090 in vivo. An E. coli construct in which the chromosomal lpxC gene is replaced by R. leguminosarum lpxC is resistant to CHIR-090 up to 100 microg/mL, or 400 times above the minimal inhibitory concentration for wild-type E. coli. Given its relatively broad spectrum and potency against diverse Gram-negative pathogens, CHIR-090 is an excellent lead for the further development of new antibiotics targeting the lipid A pathway.     

Activation of cytochrome P450 2C9-mediated metabolism: mechanistic evidence in support of kinetic observations

Studies were designed to investigate the possible mechanisms associated with the kinetic observation of CYP2C9 activation by dapsone and its phase I metabolite, N-hydroxydapsone. Kinetic studies suggested that dapsone activated CYP2C9-mediated flurbiprofen 4(')-hydroxylation by decreasing the K(m) (alpha=0.2) and increasing the V(max) (beta=1.9). Interestingly, N-hydroxydapsone also activated flurbiprofen 4(')-hydroxylation by increasing V(max) (beta=1.5) but had no effect on K(m) (alpha=0.98). To study the effects of these modulators on the binding affinity of flurbiprofen, spectral binding studies were performed. In the presence of dapsone, the spectral binding constant (K(s)) for flurbiprofen was reduced from 14.1 to 2.1 microM, while in the presence of N-hydroxydapsone, the K(s) remained unchanged (14.0 microM), which suggests that dapsone causes an increase in the affinity of flurbiprofen for CYP2C9, whereas N-hydroxydapsone does not. Additionally, stoichiometry measurements under activation conditions in the presence of dapsone resulted in a doubling of both NADPH and oxygen consumption for flurbiprofen 4(')-hydroxylation, with an overall increase in metabolite formation and a decrease in formation of peroxide and excess water. Interestingly, the presence of N-hydroxydapsone generally caused the same effects on stoichiometry as those of flurbiprofen 4(')-hydroxylation but failed to reduce excess water formation, which suggests that, while N-hydroxydapsone activates CYP2C9, it does so less efficiently and possibly through a mechanism different from that of dapsone.     

Flurbiprofen: highly potent inhibitor of prostaglandin synthesis.

Flurbiprofen, 2-(2-fluoro-4-biphenylyl)propionic acid, inhibited the formation of prostaglandin E2 from arachidonic acid by bovine seminal vesicular microsomes. It was found that flurbiprofen was an approx. 12.5-fold better inhibitor than indomethacin by comparison of their I50 values. It was suggested that the inhibition of prostaglandin synthesis by flurbiprofen might be due to the inhibition of the endoperoxygenase which catalyzed conversion of arachidonic acid to cyclic endoperoxide. Other carboxylic acid compounds such as aspirin, ibuprofen and indomethacin showed the same type of inhibition as flurbiprofen. In contrast, phenylbutazone which was a pyrozolone derivative inhibited the formation of prostaglandin E2, but not affected the endoperoxygenase reaction. The kinetic studies for inhibition of prostaglandin E2 synthetase indicated that flurbiprofen competitively inhibited prostaglandin E2 synthesis, just like indomethacin. The Ki values were estimated to be 0.128 micron for flurbiprofen and 3.18 micron for indomethacin.     

Mechanism of inhibition of dihydrofolate reductases from bacterial and vertebrate sources by various classes of folate analogues

Different classes of folate analogues have been examined with respect to the mechanism of their inhibition of dihydrofolate reductases from Escherichia coli and chicken liver. In addition, the degree of synergism between the binding of these compounds and NADPH has been investigated. Methotrexate acts as a slow, tight-binding inhibitor of both enzymes whereas trimethoprim is a slow, tight-binding inhibitor of the enzyme from E. coli and a classical inhibitor of the chicken-liver enzyme. Pyrimethamine, 2,4-diamino-6,7-dimethylpteridine, a phenyltriazine, folate and folinate exhibit classical inhibition. The degree of synergism between the binding of NADPH and the inhibitor varied from low for pyrimethamine and folate to very large for the phenyltriazine which binds to the chicken-liver enzyme almost 50 000-times more tightly in the presence of NADPH. The degree of synergism is reflected in the type of inhibition that the folate analogues yield with respect to NADPH. Compounds which exhibit slight synergism give noncompetitive inhibition whereas those with a high degree of synergism yield uncompetitive inhibition. With the exception of folinate, all compounds that act as classical inhibitors give rise to competitive inhibition with respect to dihydrofolate. Folinate exhibits competitive inhibition against NADPH and noncompetitive inhibition against dihydrofolate. These results are consistent with the formation of an enzyme-dihydrofolate-folinate complex. The (6S, alphaS)-diastereoisomer of folinate was bound at least 1000-times more tightly than the (6R, alphaS)-diastereoisomer. Consideration has been given to the possible interactions that occur between residues on the enzyme and groups on the inhibitor that give rise to slow-binding inhibition.     

Theoretical study of the electronic structure and conformation of polysilole and its derivatives

The electronic structures and conformations in the optimized geometries of polysilole and its derivatives are studied theoretically, using the one-dimensional tight-binding self-consistent field crystal orbital (SCF-CO) method. It has been found that the copolymer formed by alternating aromatic silole and quinoid thiophene has a relatively small bandgap value. We observe that polysilole may show an interesting thermochromic behavior, because the conformation of polysilole is considerably easier to change at lower temperature than that of polythiophene.     

Flurbiprofen,a unique non-steroidal anti-inflammatory drug with antimicrobial activity against Trichophyton,Microsporum and Epidermophyton species

AIMS: The objective of this study was to determine the antifungal activity of flurbiprofen against dermatophytes like Trichophyton, Microsporum and Epidermophyton species. METHODS AND RESULTS: Susceptibility tests were performed against dermatophytes like Trichophyton, Microsporum and Epidermophyton species by the microbroth dilution method. Among the dermatophytes tested, Trichophyton, Microsporum and Epidermophyton species are remarkably susceptible to this compound (MIC(50): 8-16 microg ml(-1)). A yeast pathogen, Candida albicans, and a bacterium, Staphylococcus aureus, are also susceptible to flurbiprofen. CONCLUSIONS: Flurbiprofen is a non-steroidal anti-inflammatory compound with strong antifungal activity, which is not found in two well known and medically used antifungal organic acids like benzoic and salicylic acids. SIGNIFICANCE AND IMPACT OF THE STUDY: The present action of flurbiprofen on microbes indicates its future prospects as an antimicrobial agent against dermatophytes and yeast pathogens. However, in view of the anti-inflammatory property of flurbiprofen, its antifungal action may provide an additional advantage for use as a skin ointment.     

Methotrexate, a high-affinity pseudosubstrate of dihydrofolate reductase.

Investigations have been made of the slow, tight-binding inhibition by methotrexate of the reaction catalyzed by dihydrofolate reductase from Streptococcus faecium A. Quantitative analysis has shown that progress curve data are in accord with a mechanism that involves the rapid formation of an enzyme-NADPH-methotrexate complex that subsequently undergoes a relatively slow, reversible isomerization reaction. From the Ki value for the dissociation of methotrexate from the E-NADPH-methotrexate complex (23 nM) and values of 5.1 and 0.013 min-1 for the forward and reverse rate constants of the isomerization reaction, the overall inhibition constant for methotrexate was calculated to be 58 pM. The formation of an enzyme-methotrexate complex was demonstrated by means of fluorescence quenching, and a value of 0.36 muM was determined for its dissociation constant. The same technique was used to determine dissociation constants for the reaction of methotrexate with the E-NADP and E-NADPH complexes. The results indicate that in the presence of either NADPH or NADP there is enhancement of the binding of methotrexate to the enzyme. It is proposed that methotrexate behaves as a pseudosubstrate for dihydrofolate reductase.     

On the structure of slow reacting substance of anaphylaxis: evidence of biosynthesis from arachidonic acid.

The mononuclear cells in peritoneal washings from normal rats can be induced to produce large amounts of slow reacting substance of anaphylaxis by incubation with 10 mM cysteine in the presence of the calcium ionophore A-23187. This production of slow reacting substance could be inhibited by the addition of non-steroidal anti-inflammatory drugs, e.g., indomethacin, ibuprofen and flurbiprofen, Furthermore, mediator production was inhibited by eicosatetraynoic acid, the substrate analog of arachidonic acid, and by 9,11-azoprosta-5, 13-dienoic acid (AZO analog 1), a structural analog of the prostaglandin endoperoxide, PGH2, which known to inhibit thromboxane synthesis. Relatively high concentrations of hydrocortisone acetate inhibited mediator production; this inhibition could be partly reversed by the addition of arachidonic acid or to a lesser extent by eicosatrienoic acid. Preliminary results suggest that a small fraction of the 3H-labled arachidonic acid which was taken up by these cells in vitro was associated with slow reacting substance. We postulate that slow reacting substance of anaphylaxis may be derived from a prostaglandin endoperoxide which is formed during the oxidation of arachidonic acid by the prostaglandin fatty acid cyclooxygenase.     

Purification of epidermal plasminogen activator inhibitor

A plasminogen activator inhibitor was purified from human cornified cell extract by DEAE-Sepharose, Sephacryl S-200, and high-performance liquid chromatographies on hydroxyapatite HPHT and anion-exchanger Mono Q at pH 7.2 and 8.0. The purified inhibitor showed Mr 43,000 and pI 5.2 50% inhibition of fibrinolytic activity (1.5 IU) of urokinase and tissue-type plasminogen activator was attained by 0.60 ng and 11.0 ng purified inhibitor, respectively. Synthetic substrate assay demonstrated slow tight-binding inhibition to both urokinase and tissue-type plasminogen activator. The inhibitor did not inactivate plasmin, thrombin, glandular kallikrein or trypsin.     

Structure of a murine cytoplasmic serine hydroxymethyltransferase quinonoid ternary complex: evidence for asymmetric obligate dimers

Serine hydroxymethyltransferase (SHMT) is a pyridoxal phosphate-dependent enzyme that catalyzes the reversible conversion of serine and tetrahydrofolate to glycine and methylenetetrahydrofolate. This reaction generates single carbon units for purine, thymidine, and methionine biosynthesis. The enzyme is a homotetramer comprising two obligate dimers and four pyridoxal phosphate-bound active sites. The mammalian enzyme is present in cells in both catalytically active and inactive forms. The inactive form is a ternary complex that results from the binding of glycine and 5-formyltetrahydrofolate polyglutamate, a slow tight-binding inhibitor. The crystal structure of a close analogue of the inactive form of murine cytoplasmic SHMT (cSHMT), lacking only the polyglutamate tail of the inhibitor, has been determined to 2.9 A resolution. This first structure of a ligand-bound mammalian SHMT allows identification of amino acid residues involved in substrate binding and catalysis. It also reveals that the two obligate dimers making up a tetramer are not equivalent; one can be described as "tight-binding" and the other as "loose-binding" for folate. Both active sites of the tight-binding dimer are occupied by 5-formyltetrahydrofolate (5-formylTHF), whose N5-formyl carbon is within 4 A of the glycine alpha-carbon of the glycine-pyridoxal phosphate complex; the complex appears to be primarily in its quinonoid form. In the loose-binding dimer, 5-formylTHF is present in only one of the active sites, and its N5-formyl carbon is 5 A from the glycine alpha-carbon. The pyridoxal phosphates appear to be primarily present as geminal diamine complexes, with bonds to both glycine and the active site lysine. This structure suggests that only two of the four catalytic sites on SHMT are catalytically competent and that the cSHMT-glycine-5-formylTHF ternary complex is an intermediate state analogue of the catalytic complex associated with serine and glycine interconversion.     

Kinetics of factor Xa inhibition by recombinant tick anticoagulant peptide: both active site and exosite interactions are required for a slow- and tight-binding inhibition mechanism

Recombinant tick anticoagulant peptide (rTAP) is a competitive slow- and tight-binding inhibitor of factor Xa (FXa) with a reported equilibrium dissociation constant (K(I)) of approximately 0.2 nM. The inhibitory characteristics and the high selectivity of rTAP for FXa are believed to arise from the ability of the inhibitor to specifically interact with the residues of both the active site as well as those remote from the active site pocket of the protease. To localize the rTAP-interactive sites on FXa, the kinetics of inhibition of wild-type and 18 different mutants of recombinant FXa by the inhibitor were studied by either a discontinuous assay method employing the tight-binding quadratic equation or a continuous assay method employing the slow-binding kinetic approach. It was discovered that K(I) values for the interaction of rTAP with four FXa mutants (Tyr(99) --> Thr, Phe(174) --> Asn, Arg(143) --> Ala, and a Na(+)-binding loop mutant in which residues 220-225 of FXa were replaced with the corresponding residues of thrombin) were elevated by 2-3 orders of magnitude for each mutant. Further studies revealed that the characteristic slow type of inhibition by rTAP was also eliminated for the mutants. These findings suggest that the interaction of rTAP with the P2-binding pocket, the autolysis loop, and the Na(+)-binding loop is primarily responsible for its high specificity of FXa inhibition by a slow- and tight-binding mechanism.