Inhibition of camel lens zeta-crystallin by aspirin and aspirin-like analgesics.

Camel lens zeta-crystallin was reversibly inhibited to various degrees by aspirin (acetyl salicylic acid) and the aspirin-like analgesics: paracetamol (acetaminophen) and ibuprofen (2-(4-isobutyl phenyl)-propionic acid). Among these, aspirin was the most potent inhibitor, causing nearly complete inhibition in a dose-dependent, but time-independent manner. Analysis of inhibition kinetics revealed that aspirin was uncompetitive inhibitor (K(i) 0.64 mM) with respect to NADPH and non-competitive inhibitor (K(i) 1.6 mM) with respect to the substrate, 9,10-phenanthrenequinone (PQ). Multiple-inhibition analysis showed that aspirin and pyridoxal 5' phosphate (PAL-P), a lysine specific reagent, simultaneously bound to a critical lysine residue located towards the NADPH binding region. Consistent with this, NADPH was able to substantially protect zeta-crystallin against aspirin, whereas PQ did not provide any protection. The results suggested that an essential lysine residue was the locus of aspirin binding. The inhibition of zeta-crystallin by aspirin and aspirin-like analgesics was reversible thus eliminating acetylation as a mechanism for inhibition. Reversible binding of aspirin to this lysine may cause steric hindrance resulting in uncompetitive inhibition with respect to NADPH.     

Kinetic analysis of the inhibition of the epidermal growth factor receptor tyrosine kinase by Lavendustin-A and its analogue

Lavendustin-A was reported to be a potent tyrosine kinase inhibitor of the epidermal growth factor (EGF) receptor (Onoda, T., Iinuma, H., Sasaki, Y., Hamada, M., Isshibi, K., Naganawa, H., Takeuchi, T., Tatsuta, K., and Umezawa, K. (1989) J. Nat. Prod. 52, 1252-1257). Its inhibition kinetics was studied in detail using the baculovirus-expressed recombinant intracellular domain of the EGF receptor (EGFR-IC). Lavendustin-A (RG 14355) is a slow and tight binding inhibitor of the receptor tyrosine kinase. The pre-steady state kinetic analysis demonstrates that the inhibition corresponds to a two-step mechanism in which an initial enzyme-inhibitor complex (EI) is rapidly formed followed by a slow isomerization step to form a tight complex (EI*). The dissociation constant for the initial rapid forming complex is 370 nM, whereas the overall dissociation constant is estimated to be less than or equal to 1 nM. The difference between the two values is due to the tight binding nature of the inhibitor to the enzyme in EI*. The kinetic analysis using a preincubation protocol to pre-equilibrate the enzyme with the inhibitor in the presence of one substrate showed that Lavendustin-A is a hyperbolic mixed-type inhibitor with respect to both ATP and the peptide substrate, with a major effect on the binding affinities for both substrates. An analogue of Lavendustin-A (RG 14467) showed similar inhibition kinetics to that of Lavendustin-A. The results of the pre-steady state analysis are also consistent with the proposed two-step mechanism. The dissociation constant for the initial fast forming complex in this case is 3.4 microM, whereas the overall dissociation constant is estimated to be less than or equal to 30 nM. It is a partial (hyperbolic) competitive inhibitor with respect to ATP. Its inhibition is reduced to different extents by different peptide substrates, when the peptide is added to the enzyme simultaneously with the inhibitor. When studied with the least protective peptide, K1 (a peptide containing the major autophosphorylation site of the EGF receptor), RG 14467 acts as a hyperbolic noncompetitive inhibitor with respect to the peptide.     

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.     

The interaction and inhibition of muscle lactate dehydrogenase by the alkaloid caffeine

Kinetic analysis showed that the alkaloid caffeine is a competitive inhibitor of the enzyme lactate dehydrogenase with respect to substrate pyruvate, and a non-competitive inhibitor with respect to the coenzyme NADH. The inhibitor constant Ki is 0,54 mM. Scatchard analysis determined the dissociation constant for a single independent binding site of the ternary lactate dehydrogenase - NADH - caffeine complex (KE-NADH-CAFFEINE) and the number of binding sites to be 0,14 mM and 3,83 respectively. Caffeine binds to a hydrophobic domain in the substrate binding site. Alternate nucleophilic - electrophilic functionalities within the inhibitor molecule are proposed to be the fundamental reason for the inhibition.     

The adriamycin-iron(III) complex is a potent inhibitor of protein kinase C

Using Triton X-100/lipid mixed micellar methods, we observed that the adriamycin-iron(III) complex was a potent inhibitor of protein kinase C while uncomplexed adriamycin itself was a poor inhibitor in the absence of heavy metal contaminants. The 3:1 adriamycin-iron complex was more potent than 2:1, 1:1, and 1:0 complexes. Inhibition of protein kinase C was reversible, and 50% inhibition occurred at 13 microM (adriamycin)3Fe3+. Both the catalytic and the regulatory domain of protein kinase C were affected by adriamycin-iron(III). Adriamycin-iron(III) was a competitive inhibitor of the catalytic domain of protein kinase C with respect to MgATP but not with respect to magnesium (IC50 350 microM). The predominant interaction of adriamycin-iron(III) with native protein kinase C was as a competitive inhibitor with respect to diacylglycerol. Inhibition was not competitive with respect to phosphatidylserine, calcium, magnesium, MgATP, or histone. Interaction with the regulatory domain was demonstrated by the ability of adriamycin-iron(III) to inhibit phorbol dibutyrate binding. Other adriamycin transitional metal complexes showed little inhibition of protein kinase C activity. Acetylation of the amine on the daunosamine moeity of adriamycin did not preclude the formation of a ferric complex but resulted in total loss of inhibitory activity. These results suggest that the presence of free amines in a highly structured adriamycin-iron complex is necessary for inhibition. The implications of inhibition of protein kinase C by adriamycin-iron(III) are discussed.     

Inhibition of DNA-dependent RNA polymerase from Escherichia coli by pyran copolymer

Pyran copolymer, a potent inhibitor of DNA-dependent RNA polymerase from Escherichia coli, prevented polyribonucleotide synthesis by blocking both the initiation and elongation steps. The inhibition was noncompetitive with respect to template and nucleotide triphosphate substrates. Template binding and the stability of the nascent RNA chain were not affected by the inhibitor.     

The mode of inhibition of oxidative phosphorylation by efrapeptin (A23871). Evidence for an alternating site mechanism for ATP synthesis.

Results are presented that confirm and extend earlier findings that efrapeptin is a potent inhibitor of oxidative phosphorylation. Binding of efrapeptin is shown to be reversible, and a dissociation constant for the enzyme-inhibitor complex is estimated to be 10(-8) M under conditions for either ATP synthesis or hydrolysis. Fifty per cent inhibition of the ATP hydrolysis activity of submitochondrial particles is obtained at a ratio of 0.56 mol of inhibitor/mol of enzyme. Studies of efrapeptin binding under pseudo-first order conditions show that the onset of inhibition is first order with respect to efrapeptin. Combined with the inhibition titer, these results indicate that there is one inhibitor binding site per molecule of enzyme. Steady state velocity studies using a substrate regenerating system show that efrapeptin is competitive with both ADP and phosphate during ATP synthesis. However, during ATP hydrolysis, a distinctly different mode of inhibition is indicated with respect to ATP. Data are presented which suggest that ATP promotes the binding of efrapeptin to the enzyme. Indications that efrapeptin is a catalytic site inhibitor make these results difficult to reconcile with a simple mechanistic scheme involving a single independnet catalytic site for ATP synthesis and hydrolysis. Our results are discussed in terms of support for catalytic cooperativity between adjacent subunits as recently proposed by Kayalar et al. (Kayalar, C., Rosing, J., and Boyer, P. D. (1977) J. Biol. Chem. 252, 2486-2491).     

Inhibition of p-hydroxyphenylpyruvate dioxygenase by the diketonitrile of isoxaflutole: a case of half-site reactivity

p-Hydroxyphenylpyruvate dioxygenase (HPPD) catalyzes the formation of homogentisate from p-hydroxyphenylpyruvate and molecular oxygen. In plants, this enzyme is the molecular target of new families of very active bleaching herbicides. In the study presented here, we report for the first time on the purification to homogeneity of a plant enzyme, as obtained from recombinant Escherichia coli cells expressing a cDNA encoding carrot HPPD. The purified enzyme allowed us to carry out a detailed characterization of the inhibitory properties of a diketonitile (DKN), the active inhibitor formed from the benzoylisoxazole herbicide isoxaflutole. Inhibition kinetic analyses confirmed that DKN exerts a slow and tight-binding inhibition of HPPD, competitive with respect to the p-hydroxyphenylpyruvate substrate. The stoichiometry of DKN binding to HPPD determined by kinetic analyses or by direct binding of [(14)C]DKN revealed a half-site reactivity of DKN.     

Identification and synthesis of substituted pyrrolo[2,3-d]pyrimidines as novel firefly luciferase inhibitors

A novel firefly luciferase inhibitor (3a) with a pyrrolo[2,3-d]pyrimidine core was identified in a cell-based NF-κB luciferase reporter gene assay. It potently inhibited the firefly luciferase derived from Photinus pyralis with an IC(50) value of 0.36±0.05μM. Kinetic analysis of 3a inhibition showed that it is predominantly competitive with respect to d-luciferin and uncompetitive with respect to ATP. Therefore, several pyrrolo[2,3-d]pyrimidine analogues were prepared to further investigate the structure-activity relationship (SAR) for luciferase inhibition. The most potent inhibitor of this series was 4c, which showed an IC(50) value of 0.06±0.01μM. In addition, molecular docking studies suggested that both 3a and 4c could be accommodated in the d-luciferin binding pocket, which is expected for a predominantly competitive inhibitor with respect to d-luciferin.     

Inhibition of skeletal muscle sarcoplasmic reticulum CaATPase activity by calmidazolium

Calmidazolium, a lipophilic cation and putative calmodulin-specific antagonist, inhibited potently the calcium ATPase of sarcoplasmic reticulum (SR) vesicles isolated from skeletal muscle. Based on steady-state measurements of catalytic activity over a range of MgATP, calmidazolium, and SR protein concentrations, the calculated values of the inhibition constant (KI) and binding stoichiometry were 0.06 microM and 770 nmol/mg protein, respectively. SR CaATPase inhibition apparently is not a general property of lipophilic cations since the hydrophobic anion tetraphenylboron inhibited catalysis, whereas its cationic analog, tetraphenylarsonium, did not. Enzyme inhibition by calmidazolium was noncompetitive with respect to the substrates Ca2+ and MgATP. In the presence of other SR CaATPase inhibitors, calmidazolium was competitive with respect to quercetin and noncompetitive with respect to trifluoperazine and propranolol. While calmidazolium inhibited enzyme phosphorylation by MgATP, catalysis was more sensitive to the inhibitor. Binding of calmidazolium to SR membranes produced morphological changes seen by electron microscopy as membrane thickening and loss of resolution of surface detail. Our results show that calmidazolium is a high-affinity, noncompetitive inhibitor of skeletal SR CaATPase activity, and they suggest that this inhibition is based on binding to the membrane phospholipids rather than specific antagonism of enzyme activation by calmodulin.     

The pH dependence of the inhibition of ascorbate oxidase by anions

Double-reciprocal plots of azide inhibition, with respect to ascorbate, of ascorbate oxidase indicate mixed-type inhibition at pH values above 6. This is in contrast to the simple competitive inhibition previously observed at pH 5.6. Linear replots of the slopes and intercepts of the double-reciprocal plots yield two inhibition constants. Both constants are pH dependent. Similar inhibition patterns are obtained with fluoride and thiocyanate. These results suggest the presence of two inhibitor binding sites, one of which is competitive with respect to ascorbate and the other uncompetitive.     

Inhibition of chloroplast coupling factor ATPase by 5'-p-fluorosulfonylbenzoyl adenosine.

Incubation of chloroplast coupling factor with 5′-p-fluorosulfonylbenzoyl adenosine in the 1 to 2 mM range inhibits subsequently measured ATPase activity. The inhibition is probably due to covalent binding since it survives ammonium sulfate fractionation and dialysis. The kinetics of the inhibited enzyme with respect to substrate show a decrease in V_max with no change in K_m for ATP. The presence of ATP or ADP together with the inhibitor provides some protection against inhibition. The results suggest a possible covalent attack at a nucleotide binding site, leading to inhibition of activity.     

A novel inhibitor of glycogen phosphorylase from crayfish hepatopancreas

• 1.1. A novel glycogen phosphorylase inhibitor was partially purified from crayfish hepatopancreas.
• 2.2. The inhibitor was found only in two species of crayfish examined, and not in lobster, fresh and salt water clams, mussels or cockroaches.
• 3.3. The inhibitor is a small protein (M_r = 23,000) which did not show proteolytic activity.
• 4.4. Preliminary kinetic analysis of the inhibitory mechanism indicated that it bound to both glycogen and the glycogen phosphorylase protein.
• 5.5. Inhibitor binding to glycogen resulted in a competitive inhibition pattern with respect to glycogen phosphorylase (inhibition constant of ca 10 μg/ml).
• 6.6. The inhibitor also bound glycogen phosphorylase directly with a binding coefficient of 100 μg/ml resulting in a partially non-competitive inhibition pattern with respect to phosphate.

     

Studies on the mechanism of inhibition of redox enzymes by substituted hydroxamic acids.

Substituted primary hydroxamic acids were found to inhibit the catalytic activity of a number of redox enzymes. The inhibition was not related to the nature of the metal-active site of the enzyme nor to the nature of the oxygen-containing substrate. Two easily available enzymes, mushroom tyrosinase (monophenol,dihydroyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1) and horseradish peroxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7), which were potently inhibited by hydroxamic acids, were chosen for more detailed study. A kinetic analysis of the inhibitory effects on the partially purified tyrosinase of mushroom (Agaricus bispora) revealed that inhibition was reversible and competiitive with respect to reducing substrate concentration, but was not competitive with respect to molecular oxygen concentration. A spectrophotometric and EPR study of the binding of salicylhydroxamic acid to horseradish peroxidase revealed that his hydroxamic acid was bound to the enzyme in the same manner as a typical substrate, hydroquinone. Spectroscopic and thermodynamic measurements of the binding reactions suggested that this binding site is close, to but, not directly onto, the heme group of the enzyme. From these results it is concluded that the mode of inhibition of hydroxamic acid need not be, as generally supposed, by metal chelation, and mechanisms involving either hydrogen bonding at the reducing substrate binding site or the formation of a charge transfer complex between hydroxamic acid and an electron-accepting group in the enzyme are considered to be more feasible. The relevance of these findings to deductions on the nature of other hydroxamic acid-inhibitable systems is discussed.     

Competitive, reversible, physiological? Inhibition of mitochondrial cytochrome oxidase by nitric oxide

In the mid 1990s a number of research groups recognized that mitochondrial oxygen consumption could be reversibly inhibited by nitric oxide at the level of the enzyme cytochrome c oxidase. The inhibition was apparently competitive with respect to the oxygen concentration. This review critically assesses the present state of knowledge as regards the hypothesis that nitric oxide is a competitive, reversible, physiological inhibitor of cytochrome oxidase.     

Action of alkyl cations and the natural ATPase inhibitor from mitochondria on soluble mitochondrial ATPase.

The effect of the natural ATPase inhibitor and octylguanidine on the ATPase activity of soluble oligomycin-insensitive mitochondrial F1 were compared. Both compounds induced a maximal inhibition of 60-80% in various preparation of F1 studied. The inhibition was of the uncompetitive type with respect to MgATP, and the action of the compounds was partially additive. The data suggest that octylguanidine reproduces the action of the natural ATPase inhibitor. Alkylammonium salts also affect the ATPase activity in a similar form. F1 bound to Sepharose-hexylammonium is largely inactive, whilst free hexylammonium at higher concentrations induces only a partial inhibition of the activity. This suggests that the degree of immobilization of F1 is related to the magnitude of inhibition of ATPase activity induced by alkyl cations. The binding of F1 to Sepharose-hexylammonium is prevented by high concentrations of Na+ or K+.     

Pattern of product inhibition of 5'-AMP aminohydrolase

The inhibition of 5′-AMP aminohydrolase (EC 3.5.4.6) by NH₄Cl and IMP was examined. IMP was found to be a simple competitive inhibitor with respect to the substrate, AMP, while NH₄Cl exhibited a pattern of inhibition with both noncompetitive and competitive elements. A number of possible mechanisms were analyzed. It was found that only mechanisms in which H₂O was bound subsequent to AMP binding are consistent with the data. The data are consistent with either an ordered process of binding of substrate and release of product or a ping-pong type of binding sequence. In either case, AMP binds first and IMP is the last product released. The pH dependence of NH₄Cl inhibition is consistent with the other product being NH₃.     

Inhibition of NADH-ubiquinone reductase activity by N,N'-dicyclohexylcarbodiimide and correlation of this inhibition with the occurrence of energy-coupling site 1 in various organisms

The NADH-ubiquinone reductase activity of the respiratory chains of several organisms was inhibited by the carboxyl-modifying reagent N,N'-dicyclohexylcarbodiimide (DCCD). This inhibition correlated with the presence of an energy-transducing site in this segment of the respiratory chain. Where the NADH-quinone reductase segment involved an energy-coupling site (e.g., in bovine heart and rat liver mitochondria, and in Paracoccus denitrificans, Escherichia coli, and Thermus thermophilus HB-8 membranes), DCCD acted as an inhibitor of ubiquinone reduction by NADH. By contrast, where energy-coupling site 1 was absent (e.g., in Saccharomyces cerevisiae mitochondria and Bacillus subtilis membranes), there was no inhibition of NADH-ubiquinone reductase activity by DCCD. In the bovine and P. denitrificans systems, DCCD inhibition was pseudo first order with respect to incubation time, and reaction order with respect to inhibitor concentration was close to unity, indicating that inhibition resulted from the binding of one inhibitor molecule per active unit of NADH-ubiquinone reductase. In the bovine NADH-ubiquinone reductase complex (complex I), [14C]DCCD was preferentially incorporated into two subunits of molecular weight 49,000 and 29,000. The time course of labeling of the 29,000 molecular weight subunit with [14C]DCCD paralleled the time course of inhibition of NADH-ubiquinone reductase activity.     

Dihydrodipicolinate synthase (DHDPS) from Escherichia coli displays partial mixed inhibition with respect to its first substrate, pyruvate

Dihydrodipicolinate synthase (DHDPS, E.C. 4.2.1.52) mediates the first unique reaction of (S)-lysine biosynthesis in plants and microbes-the condensation of (S)-aspartate-beta-semialdehyde ((S)-ASA) and pyruvate. It has been shown that DHDPS is partially feedback inhibited by (S)-lysine; it is suggested that this mechanism regulates flux through the DAP biosynthetic pathway. Others have characterised DHDPS from Escherichia coli with respect to (S)-lysine inhibition. They have concluded that, with respect to pyruvate, the first substrate of the reaction, DHDPS shows uncompetitive inhibition: as such, they further suggest that (S)-lysine inhibits DHDPS via interaction with the binding site for the second substrate, (S)-ASA. Yet, this finding is based on the assumption that (S)-lysine is a fully uncompetitive inhibitor. In light of crystallographic studies, which lead to the proposal that (S)-lysine affects the putative proton-relay of DHDPS, we re-evaluated the inhibition mechanism of DHDPS with respect to (S)-lysine by incorporating the observed hyperbolic inhibition. Our data showed that lysine is not an uncompetitive inhibitor, but a mixed inhibitor when pyruvate and (S)-lysine concentrations were varied. Thus, consistent with the crystallographic data, (S)-lysine must have an effect on the initial steps of the DHDPS reaction, including the binding of pyruvate and Schiff base formation.     

Nucleotide regulatory sites on skeletal myosin

Myosin Ca2+-ATPase activity decreased in the presence of ADP. Free ATP acted either as an activator or as an inhibitor depending on its concentration. The inhibition caused by ADP or ATP followed a competitive pattern with respect to the substrate. ATP, at activating concentrations, competed with dinitrophenol and with the anions SCN-, CN- and HCO3- for the same binding sites of myosin, whereas ADP did not compete with them. These results suggest that the nucleotide regulatory site or sites, different from the hydrolytic sites, seem to coincide with the anion binding sites.