Kinetic Inhibition Constants Research Articles

Quantitative continuous assay for hyaluronan synthase

A rapid, continuous, and convenient three-enzyme coupled UV absorption assay was developed to quantitate the glucuronic acid and N-acetylglucosamine transferase activities of hyaluronan synthase from Pasteurella multocida (PmHAS). Activity was measured by coupling the UDP produced from the PmHAS-catalyzed transfer of UDP-GlcNAc and UDP-GlcUA to a hyaluronic acid tetrasaccharide primer with the oxidation of NADH. Using a fluorescently labeled primer, the products were characterized by gel electrophoresis. Our results show that a truncated soluble form of recombinant PmHAS (residues 1–703) can catalyze the glycosyl transfers in a time- and concentration-dependent manner. The assay can be used to determine kinetic parameters, inhibition constants, and mechanistic aspects of this enzyme. In addition, it can be used to quantify PmHAS during purification of the enzyme from culture media.

Brain acetylcholinesterase as a marine pesticide biomarker using Brazilian fishes

Brain acetylcholinesterase (AChE) of some fishes from the coast of Rio de Janeiro State was studied as a possible pesticide biomarker in marine environmental monitoring. AChE specific activity in brain varied from 145 to 530 U/g of proteins and the Michaelis-Menten constant (K(M)) for acetylthiocholine varied from 104 to 291 microM among the 20 species studied. The enzyme sensitivity to methyl paraoxon, evaluated by the inhibition kinetic constants, shows that some species (Paralonchurus brasiliensis and Genidens genidens) are more sensitive (IC50-30 min=455 and 468 nM, respectively). The less sensitive Merluccius hubbsi and Percophis brasiliensis (IC50-30 min=3339 and 3259 nM, respectively) belong to the super-order Paracanthopterygii, which includes the more ancient species. On the other hand, more susceptible species belong to the super-order Acanthopterygii, which includes more recent species. These results suggest a possible evolutionary linkage for AChE sensitivity to methyl paraoxon. The application of inhibition kinetic constants for fish brain AChE in phylogenetic studies is still being investigated. The results have shown that a fish sentinel species should have the highest brain AChE level among the more sensitive ones.

Identification of Human P450 Isoforms Involved in the Metabolism of the Antiallergic Drug, Oxatomide, and Its Kinetic Parameters and Inhibition Constants

Oxatomide is an antiallergic drug used for the treatment of diseases mediated by type I allergy. Recently, terfenadine and astemizole, which have antiallergic actions similar to those of oxatomide, showed side effects on the cardiovascular system. This might be because concomitant drugs such as itraconazole inhibit cytochrome P450 3A4 (CYP3A4), the enzyme responsible for the degradation of terfenadine and astemizole, and thus the blood concentrations of the drugs are abnormally increased. In another article of this issue, we have reported that oxatomide is metabolized by CYP2D6-Val and CYP3A4, and simultaneously inhibits the metabolism of the model substrates for these enzymes. In this study, we performed the kinetic analysis of oxatomide metabolism using microsomes prepared from human liver, and found that the Km and Vmax values were 26.1 microM and 1254.4 pmol/mg protein/min, respectively. Ketoconazole, one of the representative inhibitors for CYP3A4, potently inhibited the metabolism of oxatomide, but other well-known CYP inhibitors did not show significant inhibition. These results suggest that the metabolism of oxatomide is principally catalyzed by CYP3A4. Furthermore, oxatomide inhibited the metabolism of (+/-) bufuralol and testosterone, model substrates for CYP2D6 and CYP3A4, respectively, in a dose-dependent manner with the Ki values of 57.4 and 24.3 microM, respectively. These observations, together with the finding that the putative highest concentration of oxatomide in blood was congruent with 40 ng/ml ( congruent with 93 nM) at 4 h after each dosage during consecutive 6-d administration, encouraged us to conclude that oxatomide won't inhibit CYP2D6 or CYP3A4 at clinical doses.

Differences in the inhibition of cytochromes P450 3A4 and 3A5 by metabolite-inhibitor complex-forming drugs.

The objectives of this study were to characterize and compare the reversible inhibition and time-dependent inactivation of cytochromes P450 3A4 and 3A5 (CYP3A4 and CYP3A5) by erythromycin, diltiazem, and nicardipine. In the following experiments, we used cDNA-expressed CYP3A Supersomes and CYP3A-phenotyped human liver microsomes. We estimated the apparent constants for reversible inhibition (Ki(app) and IC50) and the irreversible kinetic constants (KI and kinact) for time-dependent inhibition. Based on an aggregate of Ki(app) and IC50 measurements, all inhibitors showed a greater inhibitory potency for CYP3A4 compared with CYP3A5. In addition, for each inhibitor, the kinact for CYP3A4 was approximately 4-fold higher than that for CYP3A5, indicating a greater propensity for time-dependent loss of CYP3A4 activity than of CYP3A5. Difference spectra experiments revealed an NADPH-dependent peak at approximately 455 nm [metabolite-inhibitor (MI) complex] following incubation of all three drugs with CYP3A4. There was no discernable MI complex formation following CYP3A5 incubation with any of the inhibitors. However, when CYP3A4 and CYP3A5 were incubated simultaneously with erythromycin, both enzymes appeared to contribute to the formation of a MI complex. Additional experiments revealed that erythromycin caused a comparable type I spectral change when bound to CYP3A5 and CYP3A4 (Ks=48 microM and 52 microM, respectively). Moreover, CYP3A5 exhibited only a moderately slower rate for the initial N-demethylation than did CYP3A4 (intrinsic clearance=41 versus 99 microl/min/nmol, respectively). In conclusion, erythromycin, diltiazem, and nicardipine were weaker inhibitors of CYP3A5 and inactivated the enzyme at a slower rate than their respective effects on CYP3A4. With respect to erythromycin, the failure of CYP3A5 to form a MI complex appears to be the result of slowed or impaired metabolic events downstream from the initial catalytic step, possibly due to a different orientation of the substrate molecule in the active site.

General assay for sugar nucleotidyltransferases using electrospray ionization mass spectrometry

An electrospray ionization mass spectrometry-based assay has been developed to study the class of enzymes called sugar nucleotidyltransferases that couple sugar-1-phosphates and nucleotide triphosphates to form Leloir pathway glycosyl donors. The recombinant Escherichia coli and the commercially available yeast uridine-diphosphoglucose pyrophosphorylases were used as model systems. This technique allows the simultaneous and direct detection of the substrates and products without separation and, as described, is as sensitive as traditional coupled techniques. More importantly, the assay is capable of easily measuring kinetic values and inhibition constants for a range of natural and nonnatural substrates. This new assay was used to show for the first time that the reaction of the commercially available yeast uridine-diphosphoglucose pyrophosphorylase preparation is competitively inhibited by adenosine 5 ′-triphosphate (ATP), an observation that indicates a single active site that accepts both uridine 5 ′-triphosphate and ATP substrates.

Diagnosis of enzyme inhibition based on the degree of inhibition.

In this work, a method for the diagnosis of kinetic inhibition, based on the dependence of the degree of inhibition (epsilon(i)) on the inhibitor concentration [I] and on the substrate concentration [S], is presented. Because the degree of inhibition is a ratio between rates, kinetic data are normalized by the introduction of an internal control-the rate of the uninhibited reaction. Therefore, the error associated with the kinetic measurements decreases and less experimental measurements are necessary to achieve the diagnosis. The process described, which uses graphical and/or non-linear fitting procedures, allows distinguishing between 20 different kinds of inhibition, including not only linear and hyperbolic, but also parabolic and rational 2,2 inhibitions. Rational 2,2 indicates a new type of inhibition corresponding to an incomplete parabolic inhibition, i.e. mechanistically it corresponds to an inhibitor that binds to two inhibition sites producing enzymatic complexes that are still active. In spite of its comprehensiveness, the diagnosis process is greatly facilitated by the division of the diagnosis of the inhibition in a step-by-step procedure, where only two rival models are evaluated in each step. In the non-linear fittings, the choice between rival models uses a test based on information statistics theory, the Akaike information criterion test, in order to penalize complex models that tend to be favoured in fittings. Finally, equations that allow the determination of inhibition kinetic constants were also deduced. The formalism presented was tested by examining inhibition of acid phosphatase by phosphate (a linear competitive inhibitor).

Kinetic constants for the inhibition of camel retinal acetylcholinesterase by the carbamate insecticide lannate

Kinetic constants for the inhibition of camel retinal acetylcholinesterase by the carbamate insecticide lannate

Biochemical mechanisms of pyrethroid resistance in three species of mosquitoes

Esterases from insecticide resistant and susceptible Blattella germanica (L.) were examined biochemically. Two strains were utilized: Johnson Wax (JWax; susceptible), and Munsyana (MA; chlorpyrifos LD50 and LD95 resistance ratio 5.2 and 10.0). On native polyacrylamide gel electrophoresis (PAGE), MA had four visible esterase electromorphs (El, E2, E3 and E4), whereas JWax had three (El, E2 and E4). Esterases E1 and E4 were more intense in the MA strain, and none of these esterase electromorphs were acetylcholinesterases. Insecticide inhibition of native esterases within polyacrylamide gels showed an interaction of all electromorphs with the carbamate insecticide propoxur and complete inhibition of all electromorphs by the carbamate bendiocarb and the organophosphate insecticides chlorpyrifos oxon, malaoxon and paraoxon. The pyrethroid insecticides permethrin and cypermethrin had no inhibitory effects. Sequential Q-Sepharose and hydroxyapatite column chromatography was used to fractionate esterases from each strain into two groups (I and II). Following hydroxyapatite fractionation of these esterase groups, inhibition kinetic constants (ka and k3), and molecular weights were estimated. Results for ka (the rate of enzyme inhibition) indicated a greater affinity for organophosphate insecticides by MA esterases. Results for k3 (the rate of enzyme recovery) indicated lengthened times of MA esterase-inhibition by organophosphate insecticides. Therefore the role, if any, in organophosphate resistance played by MA esterases must be by sequestration. Molecular weight estimates were within the range (55–65 kDa) previously observed for esterases from both B. germanica and Culex quinquefasciatus

Examination of esterases from insecticide resistant and susceptible strains of the German cockroach, Blattella germanica (L.)

Esterases from insecticide resistant and susceptible Blattella germanica (L.) were examined biochemically. Two strains were utilized: Johnson Wax (JWax; susceptible), and Munsyana (MA; chlorpyrifos LD 50 and LD 95 resistance ratio 5.2 and 10.0). On native polyacrylamide gel electrophoresis (PAGE), MA had four visible esterase electromorphs (El, E2, E3 and E4), whereas JWax had three (El, E2 and E4). Esterases E1 and E4 were more intense in the MA strain, and none of these esterase electromorphs were acetylcholinesterases. Insecticide inhibition of native esterases within polyacrylamide gels showed an interaction of all electromorphs with the carbamate insecticide propoxur and complete inhibition of all electromorphs by the carbamate bendiocarb and the organophosphate insecticides chlorpyrifos oxon, malaoxon and paraoxon. The pyrethroid insecticides permethrin and cypermethrin had no inhibitory effects. Sequential Q-Sepharose and hydroxyapatite column chromatography was used to fractionate esterases from each strain into two groups (I and II). Following hydroxyapatite fractionation of these esterase groups, inhibition kinetic constants ( k a and k 3), and molecular weights were estimated. Results for ka (the rate of enzyme inhibition) indicated a greater affinity for organophosphate insecticides by MA esterases. Results for k3 (the rate of enzyme recovery) indicated lengthened times of MA esterase-inhibition by organophosphate insecticides. Therefore the role, if any, in organophosphate resistance played by MA esterases must be by sequestration. Molecular weight estimates were within the range (55–65 kDa) previously observed for esterases from both B. germanica and Culex quinquefasciatus

Kinetics of inactivation of aminoacylase by 2-chloromercuri-4-nitrophenol: a new type of complexing inhibitor.

The kinetic theory of the substrate reaction during modification of enzyme activity previously described [Tsou (1988) Adv. Enzymol. 61, 381-436] has been applied to a study of the inactivation kinetics of aminoacylase by 2-chloromercuri-4-nitrophenol (MNP). The results indicate that the mechanism of reaction between MNP and aminoacylase is a special type of irreversible inhibition. The main features of this type of inhibitor are as follows: (i) the reaction kinetics of inhibitor with enzyme is a single exponential process; (ii) inhibition shows a noncompetitive, complexing behavior; (iii) the first inhibitor-enzyme complex, EI, still has some enzyme activity, and hence the plot of [P]infinity versus the reciprocal of inhibitor concentration gives a straight line with a positive intercept at the ordinate. On the basis of the kinetic equation of substrate reaction in the presence of the inhibitor, a plotting method has been developed for determining the inhibition kinetic constants. As an example, all reaction kinetic constants of aminoacylase with 2-chloromercuri-4-nitrophenol have been determined. The results of the present study suggest that the essential thiol group at the active site of aminoacylase may have a significant effect on the catalytic step but is not involved in substrate binding.

Purification and characterization of fungal and mammalian phosphomannose isomerases

Phosphomannose isomerase (PMI) is essential for the production of yeast cell walls. An inhibitor which inhibits the fungal enzyme without altering the activity of the mammalian enzyme would be a potential fungicidal agent, increasingly important in view of the increasing mortality from visceral mycoses in immunosuppressed patients. We have purified human, porcine, and Candida albicans enzymes 29,000-fold to homogeneity, and characterized their physical properties, as well as their kinetic parameters, inhibition constants, and pH dependences. Surprisingly, in view of the large differences between Pseudomonas aerugenosa and Saccharomyces cerevisiae PMI, the human and C. albicans enzymes are almost identical. We suggest therefore that species-selective inhibition of the fungal rather than mammalian enzyme may require molecules which bind away from the substrate binding pocket of the enzyme.

Production of propionic acid by microbiological way. Part 2. Effect of the pH value on cell growth and acid production

AbstractThe batch propionic acid fermentation of sugar cane molasses by Propionibacterium acidi propionici PP‐1 was studied at various pH values ranging from 5 to 7. The optimum pH range for cell growth was between 6 and 7, whereas the specific growth rate decreased with the pH in the acidic range down to 0.197 h−1 at pH 5.The propionic acid yield increased with decreasing pH; it changed from 22% (wt/wt) at pH 7 to 38% at pH 5. It has been obvious that this process is inhibited by the products of the fermentation and more severely in the acidic range where the acids are in an unionized state.A generalized equation of the non‐competetive inhibition was adjusted for each pH value, and kinetic inhibition constants were estimated.

Correlation of the molecular electrostatic potential surface of an enzymatic transition state with novel transition-state inhibitors.

The transition state stabilized by nucleoside hydrolase from Crithidia fasciculata is characterized by nearly complete glycosidic bond cleavage and oxycarbonium character in the ribosyl group [Horenstein, B. A., Parkin, D. W., Estupinan, B., & Schramm, V. L. (1991) Biochemistry 30, 10788-10795]. The electrostatic potential surface of the transition state provides detailed information which should be useful in the design of transition-state analogues [Horestein, B. A., & Schramm, V. L. (1993) Biochemistry 32, 7089-7097]. The electrostatic potential surface of inosine at the transition state contains a distributed positive charge resulting from the oxycarbonium ion character of the ribosyl ring. The ribosyl ring pucker is 3'-exo as a result of the near sp2 hybridization at Cl' of the ribose ring. A series of transition-state analogues have been synthesized which incorporate single or combined features of the transition state. Each feature of the transition state was analyzed for its contribution to binding energy. Kinetic inhibition constants correlate with the similarity of the inhibitor to the experimentally determined transition-state structure. Dissociation constants for the substrate and products of the reaction of inosine, hypoxanthine, and ribose are 380, 6200, and 700 microM, respectively. A transition-state analogue was synthesized which contains the required hydroxyl groups of the ribose ring, the positive charge feature of the oxycarbonium ion, and a hydrophobic mimic of the purine ring. The inhibitor 1(S)-phenyl-1,4-dideoxy-1,4-iminoribitol acts as a competitive inhibitor with respect to inosine with a dissociation constant of 0.17 microM. In addition, the inhibitor exhibits slow-onset inhibition which provides a final equilibrium dissociation constant of approximately 0.03 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

Overall kinetic mechanism of 6-phosphogluconate dehydrogenase from Candida utilis

A complete initial velocity study of the 6-phosphogluconate dehydrogenase from Candida utilis at pH 7 and 25 degrees C in both reaction directions suggests a rapid equilibrium random kinetic mechanism with dead-end E:NADP:(ribulose 5-phosphate) and E:NADPH:(6-phosphogluconate) complexes. Like substrate-product (NADP/NADPH and 6-phosphogluconate/ribulose 5-phosphate) pairs are competitive whatever the concentration of the other substrates but noncompetitive versus the other substrates, e.g., NADPH exhibits noncompetitive inhibition versus 6-phosphogluconate. This trend also holds true for all dead-end analogs, e.g., ATP-ribose is competitive versus NADP and noncompetitive versus 6-phosphogluconate. A quantitative analysis of the kinetic inhibition constants supports the assignment of kinetic mechanism. The ratio of the maximum velocities in the oxidative decarboxylation and reductive carboxylation directions is 75.

Analysis of errors in the calculation of irreversible enzyme inhibition kinetic constants.

The kinetic constants for irreversible enzyme inhibition are determined by non-linear least-square regression using a new optimisation technique. An analysis is given of how calculation of the dissociation constant and the unimolecular rate constant is affected both by the inherent error involved in fitting an exponential curve to the plot of product concentration against time, and by the ill-conditioned nature of the equations relating these constants to the parameters of the exponential. The analysis is applied to simulated sets of product concentration curves.

Production of propionic acid by microbiological way. Part 1. Influence of the initial sugars and product concentrations

AbstractThe best yields and productivities of 0.38 g · g−1 and 0.35 g · l−1 h−1, respectively, for the propionic acid production in a batchsystem using sugar‐cane final molasses as carbon source were obtained when an initial TRS concentration of 50 g · l−1 was used.It was obvious that this process is severely inhibited by the acids produced and the most drastic effect (μ = 0) was at a TVA concentration near to 250 mmol · l−1, independently of the initial TRS concentration employed.A generalizated equation of noncompetitive inhibition was adjusted: and kinetic inhibition constants for each initial TRS concentration studied were estimated.

Glycine transport by human red blood cells and ghosts: evidence for glycine anion and proton cotransport by band 3.

Stilbene-sensitive glycine transport was investigated in human red blood cells and ghosts. We have found that this component of glycine transport was inhibited by the stilbene derivatives 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS); the apparent constant for inhibition by DNDS was 4 microM in the presence of 150 mM chloride. DNDS-sensitive glycine influx was modulated by pH such that as pH was increased from 5.9 to 9.2, transport increased from 2.5 to 140 mumol.kg Hb-1.h-1 at 37 degrees C and 100 microM glycine. The increased transport was correlated with an increase in the amount of glycine present as the anion over this pH range (0.03-40 microM glycine anion), but, in addition, pH had a direct effect on transport. Glycine influx was studied as a function of glycine anion concentration with anion varied by changing pH at a constant total glycine concentration and by changing total glycine at a constant pH. A comparison of these data demonstrated that the stilbene-sensitive glycine anion flux is stimulated by protons with half-maximal stimulation below pH 6.5 and suggests that the glycine anion and a proton are cotransported. Inorganic anions transported by band 3, including Cl, NO3, and SO4, inhibited glycine transport. Glycine flux into resealed ghosts was inhibited by Cl with an inhibition constant of 25 mM. The similarities between the kinetic constants for transport inhibition by Cl and DNDS and the kinetic constants for Cl and DNDS binding to band 3 suggest that the DNDS-sensitive glycine anion and proton cotransport is via band 3.

Chromophoric peptide substrates for the spectrophotometric assay of HIV-1 protease

Purified HIV-1 protease hydrolyzes H-Ser-Gln-Asn-Leu-Phe(NO 2)-Leu-Asp-Gly-NH 2 (Peptide 1) and acetyl-Arg-Lys-Ile-Leu-Phe(NO 2)-Leu-Asp-Gly-NH 2 (Peptide 2) between the ( p-nitro)phenylalanyl and leucyl residues. The cleavage of Peptides 1 and 2 resulted in a decrease in uv absorbance at 310 nm. The HIV-1 protease-catalyzed peptidolysis of Peptides 1 and 2 was characterized by a linear time course at substrate turnover of ≤20%. The solubilities of these substrates at pH 5.0 were sufficient to provide initial rate measurements over a concentration range of 0.05–0.5 mM. Steady-state kinetic data and inhibition constants using both spectrophotometric and high performance liquid chromatography (HPLC) analysis of the peptidolysis of these substrates resulted in comparable values.

Inhibition of soybean lipoxygenase and mouse skin tumor promotion by onion and garlic components

Onion and garlic essential oils were previously shown to inhibit mouse skin tumor promotion, as were the enzymes, lipoxygenase, and cyclooxygenase. In the present study, the inhibition of soybean lipoxygenase (EC 1.13.11.12) by onion and garlic components and related compounds was investigated. The IC50 values as well as the kinetic inhibition constants were determined for the most active compounds. Di-(1-propenyl) sulfide, an analog of the substrate moiety required for oxygenase action, was the only irreversible inhibitor observed with Ki = 59 microM and k3 = 0.53/min. Inhibition in the presence of substrate was uncompetitive at 88 and 132 microM linoleic acid with Ki = 129 microM. At 173 microM linoleic acid, however, inhibition was competitive with Ki = 66 microM. Dially trisulfide, allyl methyl trisulfide, and diallyl disulfide were competitive inhibitors, while 1-propenylpropyl sulfide and (E, Z)-4,5,9-trithiadodeca-1,6,11-triene 9-oxide (ajoene) were mixed inhibitors. Nordihydroguaiaretic acid (NDGA), the most potent lipoxygenase inhibitor, was a competitive inhibitor with Ki = 0.29 microM. The results indicate a relative potency of inhibition for structural features in the following order: di(1-propenyl) sulfide greater than an alkenyl trisulfide greater than an alkenyl disulfide. Di(n-propyl) disulfide, a major onion oil component, inhibited neither lipoxygenase nor promotion. Di(1-propenyl) sulfide and ajoene inhibited both. This suggests that the inhibition of lipoxygenase may be involved in antipromotion.

Analysis of kinetic data for irreversible enzyme inhibition.

Many organophosphorus compounds are irreversible inhibitors of acetylcholinesterase. The methods used in the literature to determine the inhibition kinetic constants usually involve either manual determination of the slope at various points along the inhibition progress curve or fitting polynomials to the curve. The present study investigates the use of non-linear-regression analysis to determine the various parameters. A method is suggested that yields accurate values for the inhibition constants under a range of circumstances.