Kinetics of the inhibition of bovine liver dihydrofolate reductase by tea catechins: origin of slow-binding inhibition and pH studies

Dihydrofolate reductase (DHFR) is the subject of intensive investigation since it appears to be the primary target enzyme for "antifolate" drugs, such as methotrexate and trimethoprim. Fluorescence quenching and stopped-flow fluorimetry show that the ester bond-containing tea polyphenols (-)-epigallocatechin gallate (EGCG) and (-)-epicatechin gallate (ECG) are potent and specific inhibitors of DHFR with inhibition constants (K(I)) of 120 and 82 nM, respectively. Both tea compounds showed the characteristics of slow-binding inhibitors of bovine liver DHFR. In this work, we have determined a complete kinetic scheme to explain the slow-binding inhibition and the pH effects observed during the inhibition of bovine liver DHFR by these tea polyphenols. Experimental data, based on fluorimetric titrations, and transient phase and steady-state kinetic studies confirm that EGCG and ECG are competitive inhibitors with respect to 7,8-dihydrofolate, which bind preferentially to the free form of the enzyme. The origin of their slow-binding inhibition is proposed to be the formation of a slow dissociation ternary complex by the reaction of NADPH with the enzyme-inhibitor complex. The pH controls both the ionization of critical catalytic residues of the enzyme and the protonation state of the inhibitors. At acidic pH, EGCG and ECG are mainly present as protonated species, whereas near neutrality, they evolve toward deprotonated species due to ionization of the ester-bonded gallate moiety (pK = 7.8). Although DHFR exhibits different affinities for the protonated and deprotonated forms of EGCG and ECG, it appears that the ionization state of Glu-30 in DHFR is critical for its inhibition. The physiological implications of these pH dependencies are also discussed.     

Kinetic and inhibition studies on reduction of diphenyl sulfoxide by guinea pig liver aldehyde oxidase

To characterize the properties of diphenyl sulfoxide (DPSO) as a new type of electron acceptor for guinea pig liver aldehyde oxidase (AO), we compared the kinetics of the reductions of DPSO and other classical electron acceptors such as O2 and ferricyanide. The double-reciprocal plot of the 2-hydroxypyrimidine (2-OH PM)-linked DPSO reduction with the highly purified enzyme was biphasic. Similar biphasic plots were obtained with the reductions of other electron acceptors. Only the lower Km value, which was obtained by extrapolation of the plot at lower concentrations of 2-OH PM, was identical with that shown by the freshly prepared crude enzyme. DPSO as well as menadione progressively inhibited the reductions of O2 and ferricyanide with time. Menadione inhibited the DPSO reduction noncompetitively with respect to 2-OH PM and competitively with respect to DPSO, while its mode of inhibition of ferricyanide reduction was uncompetitive for either the electron donor or the acceptor. On the other hand, DPSO showed an uncompetitive and a noncompetitive inhibition of ferricyanide reduction with respect to 2-OH PM and ferricyanide, respectively. These results may indicate that DPSO interacts with the enzyme at the same site as menadione, and thereby when other electron acceptors are present it serves as an actual inhibitor rather than as an electron acceptor for AO.     

Kinetic analysis of enzyme reactions with slow-binding inhibition.

In this paper we present a general kinetic study of slow-binding inhibition processes, i.e. enzyme reactions that do not respond instantly to the presence of a competitive inhibitor. The analysis that we present is based on the equation that describes the formation of products with time in each case on the experimental progress curve. It is carried out under the condition of limiting enzyme concentration and allows the discrimination between the different cases of slow-binding inhibition. The mechanism in which the formation of complex enzyme-inhibitor is a single or two slow steps or follow a rapid equilibrium, has been considered. The corresponding explicit equations of each case have been obtained and checked by numerical integration. A kinetic data analysis to evaluate the corresponding kinetic parameters is suggested. We illustrate the method, numerically by computer simulation, of the reaction and present some numerical examples that demonstrate the applicability of our procedure.     

Human placental adenosine kinase. Kinetic mechanism and inhibition

The kinetic properties of human placental adenosine kinase, purified 3600-fold, were studied. The reaction velocity had an absolute requirement for magnesium and varied with the pH. Maximal activity was observed at pH 6.5 with a Mg2+:ATP ranging from 1:1 to 2:1. High concentrations of Mg2+ or free ATP were inhibitory. Double reciprocal plots of initial velocity studies yielded intersecting lines for both adenosine and MgATP2-. The Michaelis constant was 0.4 micro M for adenosine and 75 micro M for MgATP2-. Inhibition by adenosine was observed at concentrations greater than 2.5 micro M. AMP was a competitive inhibitor with respect to adenosine and a noncompetitive inhibitor with respect to ATP. ADP was a noncompetitive inhibitor with respect to adenosine and ATP. Hyperbolic inhibition was observed during noncompetitive inhibition of adenosine kinase by AMP and ADP. Other purine and pyrimidine nucleoside mono-, di-, and triphosphates were poor inhibitors in general. S-Adenosylhomocysteine and 2'-deoxyadenosine inhibited adenosine kinase. The data suggest that (a) MgATP2- is the true substrate of adenosine kinase, and both pH and [Mg2+] may regulate its activity; (b) the kinetic mechanisms of adenosine kinase is Ordered Bi Bi; and (c) adenosine kinase may be regulated by the concentrations of its products, AMP and ADP, but is relatively insensitive to other purine and pyrimidine nucleotides.     

The kinetic mechanism of inhibition of liver pyridoxal kinase with tryptophan metabolites

The activity of purified liver pyridoxal kinase (ATP:pyridoxal 5-phosphotransferase, EC 2.7.1.35) was determined in the presence of 13 different tryptophan metabolites. Only 3-hydroxykynurenine, 3-hydroxyanthranilic acid, xanthurenic acid and quinolinic acid were found to inhibit the enzyme with I50 values of 0.1, 0.12, 0.36 and 0.42 mM, respectively. The inhibition was not related to the presence of pyridine nucleus in the metabolites molecule as was proved from the patterns of inhibition.     

Affinity labeling of pyridoxal kinase with adenosine polyphosphopyridoxal

Pyridoxal kinase is inactivated by preincubation with the affinity label reagent adenosine tetraphosphate pyridoxal (AP4-PL) at a mixing molar ratio of 5:1 AP4-PL contains structural features of the substrates pyridoxal and ATP. The substrate ATP affords substantial protection against inactivation. The extent of chemical modification by the affinity label was determined by measuring the spectroscopic properties of AP4-pyridoxyl chromophores attached to the enzyme after reduction with NaBH4. The incorporation of 2 mol of the affinity label per enzyme dimer is needed for complete inactivation of the kinase. After chymotryptic digestion of the enzyme modified with AP4-PL and reduced with tritiated NaBH4, only one radioactive peptide absorbing at 325 nm was separated by reverse-phase high performance liquid chromatography. The amino acid sequence of the radioactive peptide, elucidated by Edman degradation, revealed that a specific lysyl residue of monomeric pyridoxal kinase has reacted with the affinity label reagent. It is postulated that the modified lysyl residue is involved in direct interactions with phosphoryl groups of ATP.     

Kinetic studies on the regulation of rabbit liver pyruvate kinase

Two kinetically distinct forms of pyruvate kinase (EC 2.7.1.40) were isolated from rabbit liver by using differential ammonium sulphate fractionation. The L or liver form, which is allosterically activated by fructose 1,6-diphosphate, was partially purified by DEAE-cellulose chromatography to give a maximum specific activity of 20 units/mg. The L form was allosterically activated by K(+) and optimum activity was recorded with 30mm-K(+), 4mm-MgADP(-), with a MgADP(-)/ADP(2-) ratio of 50:1, but inhibition occurred with K(+) concentrations in excess of 60mm. No inhibition occurred with either ATP or GTP when excess of Mg(2+) was added to counteract chelation by these ligands. Alanine (2.5mm) caused 50% inhibition at low concentrations of phosphoenolpyruvate (0.15mm). The homotropic effector, phosphoenolpyruvate, exhibited a complex allosteric pattern (n(H)=2.5), and negative co-operative interactions were observed in the presence of low concentrations of this substrate. The degree of this co-operative interaction was pH-dependent, with the Hill coefficient increasing from 1.1 to 3.2 as the pH was raised from 6.5 to 8.0. Fructose 1,6-diphosphate interfered with the activation by univalent ions, markedly decreased the apparent K(m) for phosphoenolpyruvate from 1.2mm to 0.2mm, and transformed the phosphoenolpyruvate saturation curve into a hyperbola. Concentrations of fructose 1,6-diphosphate in excess of 0.5mm inhibited this stimulated reaction. The M or muscle-type form of the enzyme was not activated by fructose 1,6-diphosphate and gave a maximum specific activity of 0.3 unit/mg. A Michaelis-Menten response was obtained when phosphoenolpyruvate was the variable substrate (K(m)=0.125mm), and this form was inhibited by ATP, as well as alanine, even in the presence of excess of Mg(2+).     

Kinetic, thermodynamic and statistical studies on the inhibition of adenosine deaminase by aspirin and diclofenac

The kinetic and thermodynamic effects of aspirin and diclofenac on the activity of adenosine deaminase (ADA) were studied in 50 mM phosphate buffer pH = 7.5 at 27 and 37 degrees C, using UV-Vis spectrophotometry and isothermal titration calorimetry (ITC). Aspirin exhibits competitive inhibition at 27 and 37 degrees C and the inhibition constants are 42.8 and 96.8 microM respectively, using spectrophotometry. Diclofenac shows competitive behavior at 27 degrees C and uncompetitive at 37 degrees C with inhibition constants of 56.4 and 30.0 microM, at respectively. The binding constant and enthalpy of binding, at 27 degrees C are 45 microM, - 64.5 kJ/mol and 61 microM, - 34.5 kJ/mol for aspirin and diclofenac. Thermodynamic data revealed that the binding process for these ADA inhibitors is enthalpy driven. QSAR studies by principal component analysis implemented in SPSS show that the large, polar, planar, and aromatic nucleoside and small, aromatic and polar non-nucleoside molecules have lower inhibition constants.     

Kinetic,thermodynamic and statistical studies on the inhibition of adenosine deaminase by aspirin and diclofenac

The kinetic and thermodynamic effects of aspirin and diclofenac on the activity of adenosine deaminase (ADA) were studied in 50 mM phosphate buffer pH = 7.5 at 27 and 37°C, using UV-Vis spectrophotometry and isothermal titration calorimetry (ITC). Aspirin exhibits competitive inhibition at 27 and 37°C and the inhibition constants are 42.8 and 96.8 μM respectively, using spectrophotometry. Diclofenac shows competitive behavior at 27°C and uncompetitive at 37°C with inhibition constants of 56.4 and 30.0 μM, at respectively. The binding constant and enthalpy of binding, at 27°C are 45 μM, ? 64.5 kJ/mol and 61 μM, ? 34.5 kJ/mol for aspirin and diclofenac. Thermodynamic data revealed that the binding process for these ADA inhibitors is enthalpy driven. QSAR studies by principal component analysis implemented in SPSS show that the large, polar, planar, and aromatic nucleoside and small, aromatic and polar non-nucleoside molecules have lower inhibition constants.     

Kinetic mechanism of dihydropyrimidine dehydrogenase from pig liver

Data on initial velocity and isotope exchange at equilibrium suggest a nonclassical ping-pong mechanism for the dihydropyrimidine dehydrogenase from pig liver. Initial velocity patterns in the absence of inhibitors appeared parallel at low reactant concentration, with substrate inhibition by NADPH that is competitive with uracil and with substrate inhibition by uracil that is uncompetitive with NADPH. The Km values for both uracil (1 microM) and NADPH (7 microM) are low. As a result, it was difficult to determine whether the initial velocity pattern in the absence of added inhibitors was parallel. Thus, the pattern was redetermined in the presence of the dead-end inhibitor 2,6-dihydroxypyridine, which binds to both sites. This treatment effectively eliminates the inhibition by both substrates and increases their Km values, giving a strictly parallel pattern. Product and dead-end inhibition patterns are consistent with a mechanism in which NADPH reduces the enzyme at site 1 and electrons are transferred to site 2 to reduce uracil to dihydrouracil. The predicted mechanism is corroborated by exchange between [14C] NADP and NADPH as well as [14C]thymine and dihydrothymine in the absence of the other substrate-product pair.     

Kinetic studies of adenosine kinase from L1210 cells: a model enzyme with a two-site ping-pong mechanism

Purified adenosine kinase from L1210 cells displayed substrate inhibition by high concentrations of adenosine (Ado), ATP, and MgCl2. When incubated with ATP and MgCl2, the enzyme was phosphorylated, and the phosphorylated kinase transferred phosphate to adenosine in the absence of ATP and MgCl2. Substrate binding, isotope exchange, and kinetic studies suggested that the enzyme catalyzes the reaction by means of a two-site ping-pong mechanism with the phosphorylated enzyme as an obligatory intermediate. Among many possible pathways within this mechanism probably a random-bi ordered-bi route is the preferred sequence in which the two substrates, adenosine and MgATP, bind in a random order to form the ternary complex MgATP . E . Ado followed by the sequential dissociation of MgADP and AMP. Dissociation constants of various enzyme-substrate and enzyme-product complexes and the first-order rate constant of the rate-limiting step were estimated.     

Kinetic and structural studies of the role of the active site residue Asp235 of human pyridoxal kinase

Pyridoxal kinase catalyzes the phosphorylation of pyridoxal (PL) to pyridoxal 5′-phosphate (PLP). A D235A variant shows 7-fold and 15-fold decreases in substrate affinity and activity, respectively. A D235N variant shows ∼2-fold decrease in both PL affinity and activity. The crystal structure of D235A (2.5 Å) shows bound ATP, PL and PLP, while D235N (2.3 Å) shows bound ATP and sulfate. These results document the role of Asp235 in PL kinase activity. The observation that the active site of PL kinase can accommodate both ATP and PLP suggests that formation of a ternary Enz·PLP·ATP complex could occur in the wild-type enzyme, consistent with severe MgATP substrate inhibition of PL kinase in the presence of PLP.     

Crystal structures of human pyridoxal kinase in complex with the neurotoxins, ginkgotoxin and theophylline: insights into pyridoxal kinase inhibition

Several drugs and natural compounds are known to be highly neurotoxic, triggering epileptic convulsions or seizures, and causing headaches, agitations, as well as other neuronal symptoms. The neurotoxic effects of some of these compounds, including theophylline and ginkgotoxin, have been traced to their inhibitory activity against human pyridoxal kinase (hPL kinase), resulting in deficiency of the active cofactor form of vitamin B(6), pyridoxal 5'-phosphate (PLP). Pyridoxal (PL), an inactive form of vitamin B(6) is converted to PLP by PL kinase. PLP is the B(6) vitamer required as a cofactor for over 160 enzymatic activities essential in primary and secondary metabolism. We have performed structural and kinetic studies on hPL kinase with several potential inhibitors, including ginkgotoxin and theophylline. The structural studies show ginkgotoxin and theophylline bound at the substrate site, and are involved in similar protein interactions as the natural substrate, PL. Interestingly, the phosphorylated product of ginkgotoxin is also observed bound at the active site. This work provides insights into the molecular basis of hPL kinase inhibition and may provide a working hypothesis to quickly screen or identify neurotoxic drugs as potential hPL kinase inhibitors. Such adverse effects may be prevented by administration of an appropriate form of vitamin B(6), or provide clues of how to modify these drugs to help reduce their hPL kinase inhibitory effects.