Enzyme Intrinsic Fluorescence Research Articles

Evaluation of inhibitory effects of some novel phenolic derivatives on the mushroom tyrosinase activity: Insights from spectroscopic analyses, molecular docking and in vitro assays

Tyrosinase plays determinant role in enzymatic browning of vegetables and fresh-cut fruits. Development of new tyrosinase inhibitors is of great concern in food and agriculture. To discover new inhibitors, novel phenolic derivatives were synthesized and their inhibitory effects were investigated on activity of mushroom tyrosinase. All compounds showed potent inhibitory activities in their low concentrations and compound 4-(4-hydroxyphenyl)butan-2-one (1b) was found to be the most potent inhibitor (73.75% inhibition, IC50 value 5.6 μmol L−1). This ligand inhibited enzyme activity in a mixed pattern and kinetic parameters were also determined. In vitro assays revealed that this compound has not cytotoxicity/hemolytic effects and can be considered as safe for further investigations. Analysis of fluorescence spectra showed that all ligands quenched enzyme intrinsic fluorescence. The quenching mode and important binding parameters were also calculated. Enzyme-ligands interactions were also theoretically analyzed by molecular docking and results showed that the ligands interact with structurally/functionally critical residues.

Mycothiol/Mycoredoxin 1-dependent Reduction of the Peroxiredoxin AhpE from Mycobacterium tuberculosis

Mycobacterium tuberculosis (M. tuberculosis), the pathogen responsible for tuberculosis, detoxifies cytotoxic peroxides produced by activated macrophages. M. tuberculosis expresses alkyl hydroxyperoxide reductase E (AhpE), among other peroxiredoxins. So far the system that reduces AhpE was not known. We identified M. tuberculosis mycoredoxin-1 (MtMrx1) acting in combination with mycothiol and mycothiol disulfide reductase (MR), as a biologically relevant reducing system for MtAhpE. MtMrx1, a glutaredoxin-like, mycothiol-dependent oxidoreductase, directly reduces the oxidized form of MtAhpE, through a protein mixed disulfide with the N-terminal cysteine of MtMrx1 and the sulfenic acid derivative of the peroxidatic cysteine of MtAhpE. This disulfide is then reduced by the C-terminal cysteine in MtMrx1. Accordingly, MtAhpE catalyzes the oxidation of wt MtMrx1 by hydrogen peroxide but not of MtMrx1 lacking the C-terminal cysteine, confirming a dithiolic mechanism. Alternatively, oxidized MtAhpE forms a mixed disulfide with mycothiol, which in turn is reduced by MtMrx1 using a monothiolic mechanism. We demonstrated the H2O2-dependent NADPH oxidation catalyzed by MtAhpE in the presence of MR, Mrx1, and mycothiol. Disulfide formation involving mycothiol probably competes with the direct reduction by MtMrx1 in aqueous intracellular media, where mycothiol is present at millimolar concentrations. However, MtAhpE was found to be associated with the membrane fraction, and since mycothiol is hydrophilic, direct reduction by MtMrx1 might be favored. The results reported herein allow the rationalization of peroxide detoxification actions inferred for mycothiol, and more recently, for Mrx1 in cellular systems. We report the first molecular link between a thiol-dependent peroxidase and the mycothiol/Mrx1 pathway in Mycobacteria.

Global Fit Analysis of Glucose Binding Curves Reveals a Minimal Model for Kinetic Cooperativity in Human Glucokinase

Human pancreatic glucokinase is a monomeric enzyme that displays kinetic cooperativity, a feature that facilitates enzyme-mediated regulation of blood glucose levels in the body. Two theoretical models have been proposed to describe the non-Michaelis-Menten behavior of human glucokinase. The mnemonic mechanism postulates the existence of one thermodynamically favored enzyme conformation in the absence of glucose, whereas the ligand-induced slow transition model (LIST) requires a preexisting equilibrium between two enzyme species that interconvert with a rate constant slower than turnover. To investigate whether either of these mechanisms is sufficient to describe glucokinase cooperativity, a transient-state kinetic analysis of glucose binding to the enzyme was undertaken. A complex, time-dependent change in enzyme intrinsic fluorescence was observed upon exposure to glucose, which is best described by an analytical solution comprised of the sum of four exponential terms. Transient-state glucose binding experiments conducted in the presence of increasing glycerol concentrations demonstrate that three of the observed rate constants decrease with increasing viscosity. Global fit analyses of experimental glucose binding curves are consistent with a kinetic model that is an extension of the LIST mechanism with a total of four glucose-bound binary complexes. The kinetic model presented herein suggests that glucokinase samples multiple conformations in the absence of ligand and that this conformational heterogeneity persists even after the enzyme associates with glucose.

Structural and Functional Alterations of Catalase Induced by Acriflavine, a Compound Causing Apoptosis and Necrosis

Acriflavine is an antiseptic agent causing both apoptosis and necrosis in yeast. In this work, its effect on the structure and function of catalase, a vital enzyme actively involved in protection against oxidative stress, was investigated. In vitro kinetic studies showed that acriflavine inhibited the enzymatic activity in a competitive manner. The residual activity detectable after preincubation of catalase (1.5 nmol/L) with various concentrations of acriflavine went from 50% to 20% of the control value as the acriflavine concentration increased from 30 to 90 micromol/L. Correlatively with the decrease in activity, alterations in the enzyme's conformation were observed as indicated by fluorescence spectroscopy, circular dichroism spectroscopy, and electronic absorption spectroscopy. The enzyme's intrinsic fluorescence obtained upon excitation at either 297 nm (tryptophan residues) or 280 nm (tyrosine and tryptophan residues) decreased as a function of acriflavine concentration. Circular dichroism studies showed alterations of the protein structure by acriflavine with up to 13% decrease in alpha helix, 16% increase in beta-sheet content, 17% increase in random coil, and 4% increase in beta turns. Spectrophotometric studies showed a blueshift and modifications in the chromicity of catalase at 405 nm, corresponding to an absorbance band due to the enzyme's prosthetic group. Thus, acriflavine induced in vitro a profound change in the structure of catalase so that the enzyme could no longer function. Our results showed that acriflavine, a compound producing apoptosis and necrosis, can have a direct effect on vital functions in cells by disabling key enzymes.

Conformational changes and activity alterations induced by nickel ion in horseradish peroxidase

Conformational changes induced by the binding of nickel to horseradish peroxidase C (HRPC) were studied by electronic absorption spectroscopy, fluorescence spectroscopy and circular dichroism spectroscopy. Incubation of HRPC with various concentrations of Ni2+ for 5 minutes resulted in changes in the enzyme absorption spectrum, including variations in the intensities of the Soret, β and charge transfer (CT1) bands absorption, shift in the Soret, β and CT1 bands maxima and absorption increase at 275 nm. Increases in the enzyme's intrinsic fluorescence as determined by fluorescence spectroscopy, as well as changes in the α-helical content, as determined by circular dichroism spectroscopy, were also found. Correlatively, alterations of the enzymatic activity by Ni2+ were studied by following the H2O2-mediated oxidation of o-dianisidine and 2,2′-azinobis(3-ethylbenzothiazolinesulfonic acid) (ABTS) by HRPC. With both reducing substrates, it was found that in the presence of sufficient amount of enzyme, 1-10 mM nickel would enhance the enzymatic activity, while higher Ni2+ concentrations (20–50 mM) would inhibit it. The enzyme was completely inhibited after 5 minutes incubation in 50 mM Ni2+. Prolonged incubation would induce complete inhibition at lower Ni2+ concentrations. Spectrophotometry investigations also showed that inhibitory concentrations of Ni2+ altered compounds I and II formation, compound II being the first affected. Based on spectrophotometry, fluorescence and circular dichroism spectroscopy, and data on compounds I and II formation, a scheme is suggested for HRPC conformational changes in different Ni2+ concentrations. HRPC was found to have four potential attachment sites for Ni2+ which were sequentially occupied in a dose- and time-dependent manner by the metallic ion.

Fluorescence and FTIR study of pressure-induced structural modifications of horse liver alcohol dehydrogenase (HLADH).

The process of pressure-induced modification of horse liver alcohol dehydrogenase (HLADH) was followed by measuring in situ catalytic activity (up to 250 MPa), intrinsic fluorescence (0.1-600 MPa) and modifications of FTIR spectra (up to 1000 MPa). The tryptophan fluorescence measurements and the kinetic data indicated that the pressure-induced denaturation of HLADH was a process involving several transitions and that the observed transient states have characteristic properties of molten globules. Low pressure (< 100 MPa) induced no important modification in the catalytic efficiency of the enzyme and slight conformational changes, characterized by a small decrease in the centre of spectral mass of the enzyme's intrinsic fluorescence: a native-like state was assumed. Higher pressures (100-400 MPa) induced a strong decrease of HLADH catalytic efficiency and further conformational changes. At 400 MPa, a dimeric molten globule-like state was proposed. Further increase of pressure (400-600 MPa) seemed to induce the dissociation of the dimer leading to a transition from the first dimeric molten globule state to a second monomeric molten globule. The existence of two independent structural domains in HLADH was assumed to explain this transition: these domains were supposed to have different stabilities against high pressure-induced denaturation. FTIR spectroscopy was used to follow the changes in HLADH secondary structures. This technique confirmed that the intermediate states have a low degree of unfolding and that no completely denatured form seemed to be reached, even up to 1000 MPa.

Purine nucleoside phosphorylase from Cellulomonas sp.: physicochemical properties and binding of substrates determined by ligand-dependent enhancement of enzyme intrinsic fluorescence, and by protective effects of ligands on thermal inactivation of the enzyme

Purine nucleoside phosphorylase (PNP) from Cellulomonas sp., homotrimeric in the crystalline state, is also a trimer in solution. Other features of the enzyme are typical for “low molecular mass” PNPs, except for its unusual stability at pH 11. Purine bases, α- d-ribose-1-phosphate (R1P) and phosphate enhance the intrinsic fluorescence of Cellulomonas PNP, and hence form binary complexes and induce conformational changes of the protein that alter the microenvironment of tryptophan residue(s). The effect due to guanine (Gua) binding is much higher than those caused by other ligands, suggesting that the enzyme preferentially binds a fluorescent, most probably rare tautomeric anionic form of Gua, further shown by comparison of emission properties of the PNP/Gua complex with that of Gua anion and its N-methyl derivatives. Guanosine (Guo) and inosine (Ino) at 100 μM concentration show little and no effect, respectively, on enzyme intrinsic fluorescence, but their protective effect against thermal inactivation of the enzyme points to their forming weak binary complexes with PNP. Binding of Gua, hypoxanthine (Hx) and R1P to the trimeric enzyme is described by one dissociation constant, K d=0.46 μM for Gua, 3.0 μM for Hx, and 60 μM for R1P. The binding stoichiometry for Gua (and probably Hx) is three ligand molecules per enzyme trimer. Effects of phosphate on the enzyme intrinsic fluorescence are due not only to binding, but also to an increase in ionic strength, as shown by titration with KCl. When corrected for effects of ionic strength, titration data with phosphate are most consistent with one dissociation constant, K d=270 μM, but existence of a very weak binding site with K d>50 mM could not be unequivocally ruled out. Binding of Gua to the PNP/phosphate binary complex is weaker ( K d=1.7 μM) than to the free enzyme ( K d=0.46 μM), suggesting that phosphate helps release the purine base in the catalytic process of phosphorolysis. The results indicate that nonlinear kinetic plots of initial velocity, typical for PNPs, including Cellulomonas PNP, are not, as generally assumed, due to cooperative interaction between monomers forming the trimer, but to a more complex kinetic mechanism than hitherto considered.

Calf spleen purine nucleoside phosphorylase: complex kinetic mechanism, hydrolysis of 7-methylguanosine, and oligomeric state in solution

The active enzyme form was found to be a homotrimer, no active monomers were observed. Only in the presence of an extremely high orthophosphate concentration (0.5 M) or at a low enzyme concentration (0.2 μg/ml) with no ligands present a small fraction of the enzyme is probably in a dissociated and/or non-active form. The specific activity is invariant over a broad enzyme concentration range (0.017 μg/ml–0.29 mg/ml). At concentrations below 0.9 μg/ml and in the absence of ligands the enzyme tends to loose its catalytic activity, while in the presence of any substrate or at higher concentrations it was found to be active as a trimer. In the absence of phosphate the enzyme catalyses the hydrolysis of 7-methylguanosine (m 7Guo) with a catalytic rate constant 1.3×10 −3 s −1 as compared with the rate of 38 s −1 for the phosphorolysis of this nucleoside. The initial pre-steady-state phase of the phosphorolysis of m 7Guo, 70 s −1, is almost twice faster than the steady-state rate and indicates that the rate-limiting step is subsequent to the glycosidic bond cleavage. Complex kinetic behaviour with substrates of phosphorolytic direction (various nucleosides and orthophosphate) was observed; data for phosphate as the variable substrate with inosine and guanosine, but not with their 7-methyl counterparts, might be interpreted as two binding sites with different affinities, or as a negative cooperativity. However, the titration of the enzyme intrinsic fluorescence with 0.2 μM–30 mM phosphate is consistent with only one dissociation constant for phosphate, K d=220±120 μM. Protective effects of ligands on the thermal inactivation of the enzyme indicate that all substrates of the phosphorolytic and the synthetic reactions are able to form binary complexes with the calf spleen purine nucleoside phosphorylase. The purine bases, guanine and hypoxanthine, bind strongly with dissociation constants of about 0.1 μM, while all other ligands studied, including 7-methylguanine and 7-methylhypoxanthine, bind at least 3 orders of magnitude less potently. Binding of guanine and hypoxanthine is about 10-fold weakened by the presence of phosphate. These observations are best interpretable by the complex kinetic mechanism of the phosphorolytic reaction involving (i) random substrate binding, (ii) unusually slow, hence strongly rate-limiting, dissociation of the products guanine and hypoxanthine, but not 7-methylguanine and 7-methylhypoxanthine, and (iii) dual function of the phosphate binding site with phosphate acting as a substrate and as a modifier helping in the release of a purine base after glycosidic bond cleavage.

Interaction of 3-alkylpyridinium polymers from the sea sponge Reniera sarai with insect acetylcholinesterase.

3-Alkylpyridinium polymers (poly-APS), composed of 29 or 99 N-butyl-3-butyl pyridinium units, were isolated from the marine sponge Reniera sarai. They act as potent cholinesterase inhibitors. The inhibition kinetics pattern reveals several successive phases ending in irreversible inhibition of the enzyme. To provide more information on mechanism of inhibition, interaction of poly-APS and N-butyl-3-butyl pyridinium iodide (NBPI) with soluble dimeric and monomeric insect acetylcholinesterase (AChE) was studied by using enzyme intrinsic fluorescence and light scattering, conformational probes ANS and trypsin, and SDS-PAGE. Poly-APS quenched tryptophan fluorescence emission of AChE more extensively than NBPI. Both inhibitors exhibited a pseudo-Lehrer type of quenching. Interaction of poly-APS with dimeric AChE did not induce significant changes of the enzyme conformation as assayed by using the hydrophobic probe ANS and trypsin digestion. In contrast to NBPI, titration of both monomeric and dimeric AChE with poly-APS resulted in the appearance of large complexes detected by measuring light scattering. An excess of poly-APS produced AChE precipitation as proved on SDS-PAGE. None of the effects were observed with trypsin as a control. It was concluded that AChE aggregation and precipitation rather than the enzyme conformational changes accounted for the observed irreversible component of poly-APS inhibition.

Nonessential activation and competitive inhibition of bacterial phosphatidylinositol-specific phospholipase C by short-chain phospholipids and analogues.

Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis is an allosteric enzyme with both a phospholipid activator site and an active site. The activation of PI-PLC enzyme is optimal with phosphatidylcholine (PC) binding to the activator site and anchoring the enzyme to the interface [Zhou, C., et al. (1997) Biochemistry 36, 347-355; Zhou, C., et al. (1997) Biochemistry 36, 10089-10091]. In contrast to PC, anionic short-chain phospholipids with smaller headgroups [phosphatidylmethanol (PMe) and phosphatidic acid (PA)] as well as phosphatidylglycerol (PG) can bind to both sites playing dual roles: nonessential activation and competitive inhibition of cyclic-(1, 2)-inositol phosphate hydrolysis. PG is also a substrate, albeit a poor one, for PI-PLC, and is cleaved slowly to form alpha-glycerol phosphate. Analysis of enzyme kinetics using cIP as the substrate coupled with effects of different short-chain phospholipids on enzyme intrinsic fluorescence indicates that anionic phospholipids with small headgroups bind to the two sites with different affinities. If no interface is present, all dihexanoylphospholipids bind to the activator site more strongly than to the active site. When the activator site is occupied, it is likely that the enzyme undergoes a conformational change that allows phospholipids to bind easily to the active site. Such behavior is consistent with the observation that enzyme activation is detected at low short-chain anionic phospholipid concentrations with inhibition observed at higher concentrations, and that only inhibition is seen with these phospholipids added as monomers in the presence of a PC interface that optimally activates the PI-PLC. A kinetic model is used to extract the affinity of short-chain lipids for the active site from experimental data.

Binding of phosphate and sulfate anions by purine nucleoside phosphorylase from E. coli: ligand-dependent quenching of enzyme intrinsic fluorescence

Steady-state and time-resolved emission spectroscopy was applied to a study of the binary and ternary complexes of pure E. coli purine nucleoside phosphorylase (PNP) with phosphate (P i; a substrate) and a close non-substrate analogue (sulfate; SA). The quenching of enzyme fluorescence by P i was bimodal, best described by two modified Stern-Volmer equations fitted independently for “low” (below 0.5 mM P i) and “high” (above 0.5 mM P i) ligand concentrations. At P i > 0.5 mM, binding is characterized by a fortyfold higher dissociation constant ( K d2 = 1.12 ± 0.10 mM), i.e. by a lower affinity for phosphate, with a sevenfold lower quenching constant and 1.6-fold higher accessibility. By contrast, the binding of SA, and the resultant fluorescence quenching, was unimodal, with K d = 1.36 ± 0.07 mM, comparable to the K d for P i at “high” P i, with a total binding capacity of one sulfate or phosphate group per enzyme subunit. SA proved to be a competitive inhibitor of phosphorolysis with K i = 1.2 ± 0.2 mM vs. P i, hence similar to its K d. SA at a concentration of 5 mM did not affect the P i affinity at P i < 0.5 mM, but led to a reduced affinity and twofold higher P i binding capacities at P i > 0.5 mM. The resultant fluorescence quenching by P i decreased at 5 mM SA, with lower Stern-Volmer constant ( K SV) and fractional accessibility ( f a) values. Increasing concentrations of P i reduced the enzyme affinity for SA, characterized by a higher K d. The Hill model showed negative cooperative binding of P i in the absence and presence of 5 mM SA with Hill coefficients h = 0.60 ± 0.01 and h = 0.83 ± 0.07, respectively. SA exhibited non-cooperative binding in the absence of P i ( h = 1.08 ± 0.01) and negative cooperative binding in the presence of P i ( h < 1). PNP fluorescence decays were best fitted to a sum of two exponentials, with an average lifetime of 2.40 ± 0.14 ns, unchanged on interaction with quenching ligands, and pointing to static quenching. The overall results are relevant to the properties of PNP from various sources, in particular to the design of potent bisubstrate analogue inhibitors.

Determination of lactate by the intrinsic fluorescence of lactate oxidase

A fluorimetric method for the determination of lactate based on its enzymatic reaction with lactate oxidase (LOx) is proposed. The method relies on the relationship between the decrease in the enzyme intrinsic fluorescence during the reaction and the lactate concentration, which is shown to be the result of a primary internal filter effect on the enzyme by the pyruvate formed in the reaction. A mathematic model for predicting the effect of some parameters on the magnitude of the analytical signal was developed that provided good correlations with the experimental results. All those experimental variables affecting the analytical signal were optimized. Under the best possible conditions, the method features a linear determination range from 4 to 25 mg l lactate and a reproducibility of 3.5%, as the relative standard deviation. The proposed method allows lactate and pyruvate to be determined simultaneously. The predictions of the proposed model were also quite consistent with the experimental results obtained for synthetic samples containing both species.

Binding of substrates to human deoxycytidine kinase studied with ligand-dependent quenching of enzyme intrinsic fluorescence

Deoxycytidine kinase is a key enzyme in the salvage pathway, and its activity is required for 5'-phosphorylation of several important antiviral and cytostatic nucleoside analogues. It has recently been purified completely from human sources. Steady-state and time-resolved fluorescence of human deoxycytidine kinase was used to study its interaction with the substrates dCyd, dAdo, dUrd, dTTP, and the feedback inhibitor dCTP. Enzyme fluorescence quenching by dCTP, dCyd, dTTP, and dAdo was bimodal, and the best fits of the quenching patterns were obtained using two modified Stern-Volmer equations with two sets of quenching constants (Ksv) and accessibility values (fa) fitted independently for "low" and "high" concentration ranges of ligands. The transition between these occurred at about 20 microM dCTP, 50 microM dCyd, 30 microM dTTP, and 180 microM dAdo. Enzyme fluorescence showed unimodal quenching by dAdo and 30% reduced accessibility of the binding site in the presence of dCyd. dUrd quenching was also unimodal with Ksv = 0.0047 +/- 0.0007 microM-1 and fa = 0.75 +/- 0.05, hence in the same range as for the "high" concentration range of dAdo in the absence of dCyd, where they are 0.0025 +/- 0.0003 microM-1 and 0.73 +/- 0.03, respectively. Fluorescence quenching was used to directly determine enzyme-ligand binding and revealed bimodal binding of dCTP, dCyd, dTTP, and dAdo and unimodal binding of dUrd, and of dAdo in the presence of 0.1 microM dCyd. Transition between these two modes of binding occurred at the concentrations described above.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein structural changes accompanying formation of enzymatic transition states: tryptophan environment in ground-state and transition-state analogue complexes of adenosine deaminase.

The accessibility of protein tryptophan fluorescence to the quenching agent acrylamide has been studied in adenosine deaminase and in binary complexes of the enzyme with ground-state or transition-state analogues. Although the enzyme contains three tryptophan residues, Stern-Volmer plots are linear with all the fluorescence quenchable at high acrylamide concentrations. Tryptophan fluorescence is less easily quenched in the binary complexes than in the free enzyme, indicating a decrease in the accessibility of these residues. The greatest decrease in accessibility is found for the transition-state analogue complexes. Although the affinities of the transition-state analogues studied span a range of 10(6), the Stern-Volmer constants of the complexes are the same within experimental error. Thus, as measured by this technique, changes in enzyme conformation accompanying formation of these complexes are similar for all transition-state analogues. Resonance energy transfer from tryptophan as donor to ligand as acceptor successfully explains the differing abilities of ligands to quench the enzyme's intrinsic fluorescence upon formation of complexes in the absence of acrylamide. On the basis of Forster distance calculations, it is likely that the residues partially quenched upon formation of transition-state analogue complexes are distant from the active site.