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

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

A study on the inhibition of adenosine deaminase

Adenosine deaminase (ADA, EC 3.5.4.4) catalyses the irreversible deamination of adenosine and 2'-deoxyadenosine to inosine and 2'-deoxyinosine, respectively. In this study the inhibition of ADA from bovine spleen by several molecules with structure related to that of the substrate or product has been quantified. The inhibitors adenine, purine, inosine, 2-aminopurine, 4-aminopyrimidine, 4-aminopyridine, 4-hydroxypyridine and phenylhydrazine are shown to be competitive inhibitors with K(I) (mM) values of 0.17, 1.1, 0.35, 0.33, 1.3, 1.8, 1.4 and 0.25, respectively. Synergistic inhibition by various combinations of molecules that imitate the structure of the substrate has never been observed. Some general conclusions are: i) the enzyme ADA from bovine spleen we have used is appropriate for kinetic studies of inhibition and mechanistic studies; it can be a reference catalytic system for the homogeneous comparison of various inhibitors; ii) this enzyme presents very rigid requirements for binding the substrate: variations in the structure of adenosine imply the loss of important interactions.     

Purification and characterization of an aminopeptidase from sperm of the sea urchin, Strongylocentrotus intermedius. Ca2(+)-dependent substrate specificity as a novel feature of the enzyme

An aminopeptidase showing broad substrate specificity was purified to electrophoretic homogeneity from spermatozoa of the sea urchin, Strongylocentrotus intermedius. It is a single chain protein (Mr = 110,000) with an isoelectric point of 5.2 and shows the highest activity in a pH range between 7.0 and 7.5. Ni2+, Cu2+, Zn2+, and Hg2+, as well as 1,10-phenanthroline and p-chloromercuribenzoate, inhibit the enzyme irrespective of the substrates used, but Ca2+, Mn2+, Mg2+, and Co2+ modified the activity differently depending on the nature of the substrate. The effect of Ca2+ was most marked; it stimulated the activity toward some 4-methylcoumaryl-7-amide (MCA) substrates (for example leucine MCA), whereas it depressed the activity toward some other substrates such as arginine-MCA and lysine-MCA in a competitive manner. The rate of enzymatic hydrolysis determined for a mixture of leucine-MCA and arginine-MCA, in respect to the release of their common product (7-amino-4-methylcoumarin), was in good agreement with the value calculated on the assumption that these two substrates compete with each other for a single active site of the enzyme. Furthermore, the enzyme showed an identical Ki value for each of the competitive inhibitors examined, irrespective of the type of substrate. Ca2+ also influenced the activities toward various peptide substrates in a dual way similar to that observed on the MCA substrates. These results indicate that the sea urchin sperm aminopeptidase has an active site that alters its substrate preference depending on the Ca2+ concentration of the reaction medium.     

A study on the inhibition of adenosine deaminase

Adenosine deaminase (ADA, EC 3.5.4.4) catalyses the irreversible deamination of adenosine and 2′-deoxyadenosine to inosine and 2′-deoxyinosine, respectively. In this study the inhibition of ADA from bovine spleen by several molecules with structure related to that of the substrate or product has been quantified. The inhibitors adenine, purine, inosine, 2-aminopurine, 4-aminopyrimidine, 4-aminopyridine, 4-hydroxypyridine and phenylhydrazine are shown to be competitive inhibitors with K_I (mM) values of 0.17, 1.1, 0.35, 0.33, 1.3, 1.8, 1.4 and 0.25, respectively. Synergistic inhibition by various combinations of molecules that imitate the structure of the substrate has never been observed. Some general conclusions are: i) the enzyme ADA from bovine spleen we have used is appropriate for kinetic studies of inhibition and mechanistic studies; it can be a reference catalytic system for the homogeneous comparison of various inhibitors; ii) this enzyme presents very rigid requirements for binding the substrate: variations in the structure of adenosine imply the loss of important interactions.     

Kinetic mechanism of the endogenous lactate dehydrogenase activity of duck epsilon-crystallin

Initial velocity, product inhibition, and substrate inhibition studies suggest that the endogenous lactate dehydrogenase activity of duck epsilon-crystallin follows an order Bi-Bi sequential mechanism. In the forward reaction (pyruvate reduction), substrate inhibition by pyruvate was uncompetitive with inhibition constant of 6.7 +/- 1.7 mM. In the reverse reaction (lactate oxidation), substrate inhibition by L-lactate was uncompetitive with inhibition constant of 158 +/- 25 mM. The cause of these inhibitions may be due to epsilon-crystallin-NAD(+)-pyruvate and epsilon-crystallin-NADH-L-lactate abortive ternary complex formation as suggested by the multiple inhibition studies. Pyruvate binds to free enzyme very poorly, with a very large dissociation constant. Bromopyruvate, fluoropyruvate, pyruvate methyl ester, and pyruvate ethyl ester are alternative substrates for pyruvate. 3-Acetylpyridine adenine dinucleotide, nicotinamide 1,N6-ethenoadenine dinucleotide, and nicotinamide hypoxanthine dinucleotide serve as alternative coenzymes for epsilon-crystallin. All the above alternative substrates or coenzymes showed an intersecting initial-velocity pattern conforming to the order Bi--Bi kinetic mechanism. Nicotinic acid adenine dinucleotide, thionicotinamide adenine dinucleotide, and 3-aminopyridine adenine dinucleotide acted as inhibitors for this enzymatic crystallin. The inhibitors were competitive versus NAD+ and noncompetitive versus L-lactate. alpha-NAD+ was a noncompetitive inhibitor with respect to the usual beta-NAD+. D-Lactate, tartronate, and oxamate were strong dead-end inhibitors for the lactate dehydrogenase activity of epsilon-crystallin. Both D-lactate and tartronate were competitive inhibitors versus L-lactate while oxamate was a competitive inhibitor versus pyruvate. We conclude that the structural requirements for the substrate and coenzyme of epsilon-crystallin are similar to those of other dehydrogenases and that the carboxamide carbonyl group of the nicotinamide moiety is important for the coenzyme activity.     

Steady-state inhibitory kinetic studies on the ligand binding modes of Aspergillus niger glucoamylase.

Inhibitory activities of 1-deoxynojirimycin and gluconolactone on Aspergillus niger glucoamylase were studied in relation to the subsite structure of the enzyme. Although both of these inhibitors are considered to bind at subsite 1 of the enzyme active site, 1-deoxynojirimycin showed competitive type inhibition but gluconolactone was a mixed type (or noncompetitive type) inhibitor for the hydrolysis of p-nitrophenyl alpha-D-glucoside. The former type of inhibition suggested that the main binding mode of the substrate was productive, but the latter, nonproductive. A possible way of explaining these apparent inconsistent results is to assume that the main binding mode of the substrate is productive and gluconolactone forms a nonproductive ternary complex with the enzyme and the substrate.     

Irreversible inhibition of the thermophilic esterase EST2 from <Emphasis Type="Italic">Alicyclobacillus acidocaldarius</Emphasis>

Kinetic studies of irreversible inhibition in recent years have received growing attention owing to their relevance to problems of basic scientific interest as well as to their practical importance. Our studies have been devoted to the characterization of the effects that well-known acetylcholinesterase irreversible inhibitors exert on a carboxylesterase (EST2) from the thermophilic eubacterium Alicyclobacillus acidocaldarius. In particular, sulfonyl inhibitors and the organophosphorous insecticide diethyl-p-nitrophenyl phosphate (paraoxon) have been studied. The incubation of EST2 with sulfonyl inhibitors resulted in a time-dependent inactivation according to a pseudo-first-order kinetics. On the other hand, the EST2 inactivation process elicited by paraoxon, being the inhibition reaction completed immediately after the inhibitor addition, cannot be described as a pseudo-first-order kinetics but is better considered as a high affinity inhibition. The values of apparent rate constants for paraoxon inactivation were determined by monitoring the enzyme/substrate reaction in the presence of the inhibitor, and were compared with those of the sulfonyl inhibitors. The protective effect afforded by a competitive inhibitor on the EST2 irreversible inhibition, and the reactivation of a complex enzyme/irreversible-inhibitor by hydroxylamine and 2-PAM, were also investigated. The data have been discussed in the light of the recently described dual substrate binding mode of EST2, considering that the irreversible inhibitors employed were able to discriminate between the two different binding sites.     

Inhibition studies of soybean trypsin-like enzyme

The results of inhibition studies of soybean trypsin-like enzyme (STLE) by substrate analogues (derivative of arginine) suggested that a net negative charge exists at or near the substrate binding region of the enzyme. On hydrolysis of substrates, this negative charge seems to repel the products from the substrate binding region and facilitate the turn-over of substrates. From the data on inhibition by various amidines, guanidines, and amines, some information about the structure of the hydrophobic binding pocket of STLE was obtained. The inactivation of STLE by irreversible inhibitors, diisopropylfluorophosphate (DFP) and tosyl-lysine chloromethyl ketone (Tos-Lys-CH2Cl), was decreased by competitive inhibitors. This means that these irreversible inhibitors bind with residues at the substrate binding region, probably serine and histidine residues, respectively.     

Design and evaluation of Trypanosoma brucei metacaspase inhibitors

Metacaspase (MCA) is an important enzyme in Trypanosoma brucei, absent from humans and differing significantly from the orthologous human caspases. Therefore MCA constitutes a new attractive drug target for antiparasitic chemotherapeutics, which needs further characterization to support the discovery of innovative drug candidates. A first series of inhibitors has been prepared on the basis of known substrate specificity and the predicted catalytic mechanism of the enzyme. In this Letter we present the first inhibitors of TbMCA2 with low micromolar enzymatic and antiparasitic activity in vitro combined with low cytotoxicity.     

Mechanistic and kinetic studies of inhibition of enzymes

A graphical method for analyzing enzyme data to obtain kinetic parameters, and to identify the types of inhibition and the enzyme mechanisms, is described. The method consists of plotting experimental data as nu/(V0 - nu) vs 1/(I) at different substrate concentrations. I is the inhibitor concentration; V0 and nu are the rates of enzyme reaction attained by the system in the presence of a fixed amount of substrate, and in the absence and presence of inhibitor, respectively. Complete inhibition gives straight lines that go through the origin; partial inhibition gives straight lines that converge on the 1-I axis, at a point away from the origin. For competitive inhibition, the slopes of the lines increase with increasing-substrate concentration; with noncompetitive inhibition, the slopes are independent of substrate concentration; with uncompetitive inhibition, the slopes of the lines decrease with increasing substrate concentrations. The kinetic parameters, Km, Ki, Ki', and beta (degree of partiality) can best be determined from respective secondary plots of slope and intercept vs substrate concentration, for competitive and noncompetitive inhibition mechanism or slope and intercept vs reciprocal substrate concentration for uncompetitive inhibition mechanism. Functional consequencs of these analyses are represented in terms of specific enzyme-inhibitor systems.     

Elucidating substrate and inhibitor binding sites on the surface of GSK-3β and the refinement of a competitive inhibitor

A molecular understanding of substrate recognition of protein kinases provides an important basis for the development of substrate competitive inhibitors. Here, we explored substrate recognition and competitive inhibition of glycogen synthase kinase (GSK)-3β using molecular and computational tools. In previous work, we described Gln89 and Asn95 within GSK-3β as important substrates binding sites. Here, we show that the cavity bordered by loop 89-QDKRFKN-95, located in the vicinity of the GSK-3β catalytic core, is a promiscuous substrate binding subsite. Mutations within this segment highlighted Phe93 as an additional essential contact residue for substrates' recognition. However, unlike Gln89 and Asn95, Phe93 was also important for the binding of our previously described substrate competitive inhibitor, L803 [KEAPPAPPQS(p)P], and its cell-permeable variant L803-mts. The effects of the substitution of charged or polar residues within L803 further suggested that binding to GSK-3β is governed by hydrophobic interactions. Our computational model of GSK-3β bound to L803 was in agreement with the experimental data. It revealed L803 binding with a hydrophobic surface patch and identified interactions between Pro8 (L803) and Phe93 (GSK-3β). Computational modeling of new L803 variants predicted that inhibition would be strengthened by adding contacts with Phe93 or by increasing the hydrophobic content of the peptide. Indeed, the newly designed L803 variants showed improved inhibition. Our study identified different and overlapping elements in GSK-3β substrate and inhibitor recognition and provides a novel example for model-based rational design of substrate competitive inhibitors for GSK-3.     

Calculation of inhibitor Ki and inhibitor type from the concentration of inhibitor for 50% inhibition for Michaelis-Menten enzymes

The use of I50 (concentration of inhibitor required for 50% inhibition) for enzyme or drug studies has the disadvantage of not allowing easy comparison among data from different laboratories or under different substrate conditions. Modifications of the Michaelis-Menten equation for treatment of inhibitors can allow both the determination of the type of inhibition (competitive, noncompetitive, and uncompetitive) and the Ki for the inhibitor. For competitive and uncompetitive inhibitors when the assay conditions are [S] = Km, then Ki = I50/2. For different conditions of [S] there is a divergence between competitive and uncompetitive inhibitors that may be used to identify the type of inhibitor. The equation for Ki also differs. For noncompetitive inhibitors the Ki = I50 and this relationship is valid with changing [S]. The equations developed require a single substrate, reversible-type inhibitors, and kinetics of the Michaelis-Menten type. Examples of the use of the equations are illustrated with experimental data from scientific publications.     

P-Glycoprotein (P-gp) expressed in a confluent monolayer of hMDR1-MDCKII cells has more than one efflux pathway with cooperative binding sites

The multidrug resistance transporter P-glycoprotein (P-gp) effluxes a wide range of substrates and can be affected by a wide range of inhibitors or modulators. Many studies have presented classifications for these binding interactions, within either the context of equilibrium binding or the Michaelis-Menten enzyme analysis of the ATPase activity of P-gp. Our approach is to study P-gp transport and its inhibition using a physiologically relevant confluent monolayer of hMDR1-MDCKII cells. We measure the elementary rate constants for P-gp efflux of substrates and study inhibition using pairwise combinations with a different unlabeled substrate acting as the inhibitor. Our current kinetic model for P-gp has only a single binding site, because a previous study proved that the mass-action kinetics of efflux of a single substrate were not sensitive to whether there are one or more substrate-binding and efflux sites. In this study, using this one-site model, we found that, with "high" concentrations of either a substrate or an inhibitor, the elementary rate constants fitted independently for each of the substrates alone quantitatively predicted the efflux curves, simply applying the assumption that binding at the "one site" was competitive. On the other hand, at "low" concentrations of both the substrate and inhibitor, we found no inhibition of the substrate efflux, despite the fact that both the substrate and inhibitor were being well-effluxed. This was not an effect of excess "empty" P-gp molecules, because the competitive efflux model takes site occupancy into account. Rather, it is quantitative evidence that the substrate and inhibitor are being effluxed by multiple pathways within P-gp. Remarkably, increasing the substrate concentration above the "low" concentration, caused the inhibition to become competitive; i.e., the inhibitor became effective. These data and their analysis show that the binding of these substrates must be cooperative, either positive or negative.     

Effects of Oxodiperoxovanadate (V) Complexes on the Activity of Green Crab (Scylla serrata) Alkaline Phosphatase

Green crab (Scylla serrata) alkaline phosphatase (EC 3.1.3.1) is a metalloenzyme that catalyzes the nonspecific hydrolysis of phosphate monoesters. The effects of some pollutants in seawater on the activity of the enzyme will result in the loss of the biological function of the enzyme, which will affect the exuviating crab shell and threaten the survival of the animal. In the present paper, the effects of four oxodiperoxovanadate (V) complexes on the activity of green crab alkaline phosphatase have been studied. The results show that these vanadate derivatives can lead to reversible inactivation. The equilibrium constants for binding of inhibitors with the enzyme and/or the enzyme–substrate complexes have been determined. The results show that sodium (2,2'-bipyridine)oxodiperoxovanadate, pV(bipy), and potassium oxodiperoxo-(1,10-phenanthroline)vanadate, pV(phen), are competitive inhibitors, while potassium picolinato-oxodiperoxo-vanadate, pV(pic), and oxalato-oxodiperoxovanadate, pV(ox), are mixed-type inhibitors. These results suggest that pV(bipy) is a considerably more potent competitive inhibitor than pV(phen) and that the competitive inhibition effect of pV(pic) is stronger than that of pV(ox), but the non-competitive inhibition effect of pV(ox) is stronger than that of pV(pic).     

Purine and pyrimidine inhibitors of arginase.

1. Adenosine, inosine, adenine and uric acid are competitive inhibitors and cytidine and cytosine noncompetitive inhibitors of bovine liver arginase (L-arginine amidinohydrolase, EC 3.5.3.1). 2. The affinity of the enzyme for these inhibitors was 10--100 times as great as for substrate in terms of Ki versus Km. 3. These nucleic acid metabolites may thus function in vivo to regulate the urea cycle. 4. Several naturally occuring competitive and noncompetitive inhibitors of arginase of unknown structure have been isolated from plant and animal tissue. From their properties and methods of isolation, they may be the purines and pyrimidines herein described. 5. These purines and pyrimidines have no effect on tryptic hydrolysis.     

N-acetylglucosaminono-1,5-lactone oxime and the corresponding (phenylcarbamoyl)oxime. Novel and potent inhibitors of beta-N-acetylglucosaminidase

Using N-acetylglucosaminono-1,5-lactone (1) as the reference, the inhibitory activity of its (formal) derivatives N-acetylglucosaminono-1,5-lactone oxime (2) and N-acetylglucosaminono-1,5-lactone O-(phenylcarbamoyl)-oxime (3) was tested against beta-N-acetylglucosaminidase of different origins (animal, plant, fungus). Displaying inhibition constants of 0.45 microM and 0.62 microM, for the animal and plant enzyme, respectively, the simple oxime 2 was about equally potent as the parent lactone 1, and 50-400 times more efficient than two recently described new beta-N-acetylglucosaminidase inhibitors. The (phenylcarbamoyl)oxime 3 performed even better, particularly with the fungal enzyme (Ki = 40 nM), i.e. was about 350 times more potent than the lactone. In all cases competitive inhibition was observed with 4-nitrophenyl-beta-N-acetylglucosaminide as the substrate. With Ki/Km ratios up to 3300 for 2 and 13,600 for 3, the mode of action of these novel inhibitors is probably that of transition state mimicry. Suggestions are made for their use as a tool in biological research.     

Alpha-hydroxytropolones are noncompetitive inhibitors of human RNase H1 that bind to the active site and modulate substrate binding

The ribonucleases H (RNases H) of HIV and hepatitis B virus are type 1 RNases H that are promising drug targets because inhibiting their activity blocks viral replication. Eukaryotic ribonuclease H1 (RNase H1) is an essential protein and a probable off-target enzyme for viral RNase H inhibitors. α-hydroxytropolones (αHTs) are a class of anti-RNase H inhibitors that can inhibit the HIV, hepatitis B virus, and human RNases H1; however, it is unclear how these inhibitors could be developed to distinguish between these enzymes. To accelerate the development of selective RNase H inhibitors, we performed biochemical and kinetic studies on the human enzyme, which was recombinantly expressed in Escherichia coli. Size-exclusion chromatography showed that free RNase H1 is monomeric and forms a 2:1 complex with a substrate of 12 bp. FRET heteroduplex cleavage assays were used to test inhibition of RNase H1 in steady-state kinetics by two structurally diverse αHTs, 110 and 404. We determined that turnover rate was reduced, but inhibition was not competitive with substrate, despite inhibitor binding to the active site. Given the compounds’ reversible binding to the active site, we concluded that traditional noncompetitive and mixed inhibition mechanisms are unlikely. Instead, we propose a model in which, by binding to the active site, αHTs stabilize an inactive enzyme–substrate–inhibitor complex. This new model clarifies the mechanism of action of αHTs against RNase H1 and will aid the development of RNase H inhibitors selective for the viral enzymes.     

Purine nucleoside phosphorylase of rabbit liver. Mechanism of catalysis.

Initial velocity studies and product inhibition patterns for purine nucleoside phosphorylase from rabbit liver were examined in order to determine the predominant catalytic mechanism for the synthetic (forward) and phosphorolytic (reverse) reactions of the enzyme. Initial velocity studies in the absence of products gave intersecting or converging linear double reciprocal plots of the kinetic data for both the synthetic and phosphorolytic reactions of the enzyme. The observed kinetic pattern was consistent with a sequential mechanism, requiring that both substrates add to the enzyme before products may be released. The product inhibition patterns showed mutual competitive inhibition between guanine and guanosine as variable substrates and inhibitors. Ribose 1-phosphate and inorganic orthophosphate were also mutually competitive toward each other. Other combinations of substrates and products gave noncompetitive inhibition. Apparent inhibition constants calculated for guanine as competitive inhibitor and for ribose 1-phosphate as noncompetitive inhibitor of the enzyme, with guanosine as variable substrate, did not vary significantly with increasing concentrations of inorganic orthophosphate as fixed substrate. These results suggest that the mechanism was order and that substrates add to the enzyme in an obligatory order. Dead end inhibition studies carried out in the presence of the products guanine and ribose 1-phosphate, respectively, showed that the kinetically significant abortive ternary complexes of enzyme-guanine-inorganic orthophosphate (EQB) and enzyme-guanose-ribose 1-phosphate (EAP) are formed. The results of dead end inhibition studies are consistent with an obligatory order of substrate addition to the enzyme. The nucleoside or purine is probably the first substrate to form a binary complex with the enzyme, and with which inorganic orthophosphate or ribose 1-phosphate may interact as secondary substrates. The evidences presented in this investigation support an Ordered Theorell-Chance mechanism for the enzyme.     

A new class of potent reversible inhibitors of metallo-proteinases: C-terminal thiol-peptides as zinc-coordinating ligands

A number of substrate analogous peptides containing a phosphoramidate, phosphonate ester, hydroxamate, carboxylate or sulfhydryl group are known to be inhibitors of thermolysin and other metalloproteinases. According to the specificity, most of the inhibitors mimic the prime site of the active center. Hitherto, peptidyl derivatives with a thiol group at the C-terminus have not been described. We have synthesized the protected cysteamides Ac-Ala-Ala-CA-SH and Z-Aa1-Aa2-CA-SH (Aa1: Ala, Pro; Aa2: Ala, Leu). The binding of these thiol peptide inhibitors to the metalloproteinases is characterized first by the coordination of the thiolate group of the inhibitor to the catalytic zinc ion and second by the subsite interaction of the peptide ligand in the active site of the enzyme. All peptide derivatives were competitive inhibitors of the zinc metalloproteinase thermolysin. The strongest inhibition was found with Z-Pro-Leu-CA-SH (Ki = 30 microM). Substitution of the N-protecting benzyloxycarbonyl residue towards the acetyl group in the peptide inhibitor, the inhibition constant decreased about 25 times.     

Determination of rate constants for the irreversible inhibition of acetylcholine esterase by continuously monitoring the substrate reaction in the presence of the inhibitor

The kinetics of the irreversible inhibition of acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) by diisopropyl fluorophosphate and paraoxon have been studied by the approach of following the substrate reaction continuously in the presence of both the substrate and the inhibitor based on kinetic equations previously derived (Tsou, C.-L. (1965) Acta Biochim. Biophys. Sinica 5, 387-417). From determinations of the effects of different concentrations of substrate and the inhibitors on the apparent rate constants for the irreversible inhibition reactions it can be shown that these inhibitors are of the competitive complexing type. Both the reversible dissociation constant for the enzyme inhibitor complex and the rate constant for the subsequent phosphorylation step can be obtained from suitable plots of the experimental data.