Inactivation of 1-Aminocyclopropane-1-Carboxylate Synthase by l-Vinylglycine as Related to the Mechanism-Based Inactivation of the Enzyme by S-Adenosyl-l-Methionine

The pyridoxal phosphate-dependent 1-aminocyclopropane-1-carboxylate (ACC) synthase catalyzes the conversion of S-adenosyl-l-methionine (AdoMet) to ACC, and is inactivated by AdoMet during the reaction. l-Vinylglycine was found to be a competitive inhibitor of the enzyme, and to cause a time-dependent inactivation of the enzyme. The inactivation required the presence of pyridoxal phosphate and followed pseudo-first-order kinetics at various concentrations of l-vinylglycine. The Michaelis constant for l-vinylglycine in the inactivation reaction (K_inact) was 3.3 millimolar and the maximum rate constant (k_max) was 0.1 per minute. These findings, coupled with the previous observations that the suicidal action of AdoMet involved a covalent linkage of the aminobutyrate portion of AdoMet to the enzyme, support the view that the mechanism-based inactivation of ACC synthase by the substrate AdoMet proceeds through the formation of a vinylglycine-ACC synthase complex as an intermediate.     

Inhibition of extracellular lipase from Streptomyces rimosus with 3,4-dichloroisocoumarin

Kinetic characterization of lipase inhibition was performed by activity measurement and mass spectrometry (MS), for the first time with serine-protease inhibitor 3,4-dichloroisocoumarin (DCI). Inhibition of Streptomyces rimosus extracellular lipase (SrLip), a member of the SGNH superfamily, by means of DCI follows the mechanism of two-step irreversible inhibition. The dissociation constant of the noncovalent E?I complex and first-order rate constant for inactivation were determined by incubation (K_i* = 26.6?±?2.8 µM, k₂ = 12.2?±?0.6 min–1) or progress curve (K_i* = 6.5?±?1.5 µM, k₂ = 0.11?±?0.01 min–1) method. Half-times of reactivation for lipase inhibited with 10-fold molar excess of DCI were determined by activity measurement (t_1/2 = 11.3?±?0.2?h), matrix-assisted laser desorption/ionization (MALDI, t_1/2 = 13.5?±?0.4?h), and electro-spray ionization (ESI, t_1/2 = 12.2?±?0.5?h) MS. The active SrLip concentration was determined by incubating the enzyme with near equimolar concentrations of DCI, followed by activity and MS measurement.     

Species Differences in the Selective Inhibition of Monoamine Oxidase (1-methyl-2-phenylethyl)hydrazine and its Potentiation by Cyanide

The potentiating effects of cyanide on the inhibition of rat liver mitochondrial monoamine oxidase-A & B and of ox liver mitochondrial MAO-B by pheniprazine [(1-methyl-2-phenylethyl)hydrazine] has been studied. Pheniprazine was shown to behave as a mechanism-based MAO inhibitor. For rat liver MAO-B, the initial non-covalent step was characterized by dissociation constant (K _i) of 2450 nM and the first-order rate constant (k ₊₂) for the covalent adduct formation was 0.16 min⁻¹. As a reversible inhibitor it was selective towards rat liver MAO-A (K _i = 420 nM) but the rate of irreversible inhibition of that enzyme was considerably slower (k ₊₂ = 0.06 min⁻¹). MAO-B from ox liver more closely resembled MAO-A from the rat in sensitivity to reversible inhibition by pheniprazine (K _i = 450 nm) but it was closer to rat liver MAO-B in rate of irreversible inhibition (k ₊₂ = 0.29 min⁻¹). The K _i values were significantly decreased in the presence of KCN but there was little effect on the k ₊₂ values. However, sensitivities of the different enzymes to KCN varied widely and considerably higher concentrations of KCN were required for this effect to be apparent with the rat liver mitochondrial MAO-A than with MAO-B from rat and ox liver. The kinetic behaviour of cyanide activation was consistent with partial (non-essential) competitive activation in all cases. Special issue dedicated to Dr. Moussa Youdim.     

Inhibition of phosphate uptake in barley roots by hydroxy-benzoic acids

The influence of 15 hydroxy-benzoic acids upon active inorganic phosphate absorption by barley roots was examined. For each compound an inhibition constant (k_i) was determined, i.e. the concentration of compound required to bring about a 50% inhibition of absorption. The k_i values of the benzoic acids were strongly correlated with their octanol—water partition coefficients and their pK_a values. This suggests that the inhibition of normal membrane functions, brought about by benzoic acids, results from a generalized increase in cell membrane permeability. Salicylate derivatives were generally more inhibitory than would be predicted from their partition coefficients; their pronounced toxicity probably arises from structural impediments to their detoxication.     

Reversible Inhibition of Enzymatic Systems Involving Covalent Intermediates

Abstract

Reversible inhibition phenomena are analyzed for enzymatic systems involving covalent intermediates, where the inhibitor can bind to the pure enzyme, the Henri-Michaelis complex or the covalent intermediate, or to two or three of these enzyme forms. Classical competitive, non-competitive or un-competitive phenomena can be observed in some cases but unexpected features are also observed. Complex phenomena sometimes prevail where increased k_cat/K_m values can be accompanied by decreased or increased k_cat values.     

Oxidative inactivation of the lymphoid tyrosine phosphatase mediated by both general and active site directed NO donors

Oxidative modification of protein tyrosine phosphatases (PTPs) has recently been recognized as an important regulatory mechanism in biological systems. Reported herein is the oxidative inactivation of the lymphoid tyrosine phosphatase (LYP) with both the general nitrosating reagent sodium nitroprusside (SNP) and also a novel peptide-based nitrosating reagent, Ac-ARLIEDNE(HcyNO)TAREG-NH₂, where HcyNO = S-nitrosohomocysteine. The SNP oxidatively inactivated LYP with a k_inact of 0.383 per min and a K_I of 27.4 μM and mixed-type inactivation kinetics. The peptide was a competitive LYP inactivator with a k_inact of 0.0472 per min and a K_I of 7.00 μM. LYP nitrosation by SNP was characterized by the addition of several NO moieties to the enzyme, while oxidation of LYP by the peptide did not result in the formation of a LYP-NO adduct. We propose that general NO donors promiscuously nitrosate any free cysteine residue while the active-site directed peptide selectively oxidizes the catalytic cysteine residue, resulting in the formation of a disulfide bond between the catalytic cysteine residue and a second cysteine in the active site.     

Biochemical characterization of tirabrutinib and other irreversible inhibitors of Bruton's tyrosine kinase reveals differences in on - and off - target inhibition

BackgroundBruton's tyrosine kinase (BTK) is a key component of the B-cell receptor (BCR) pathway and a clinically validated target for small molecule inhibitors such as ibrutinib in the treatment of B-cell malignancies. Tirabrutinib (GS-4059/ONO-4059) is a selective, once daily, oral BTK inhibitor with clinical activity against many relapsed/refractory B-cell malignancies.MethodsCovalent binding of tirabrutinib to BTK Cys-481 was assessed by LC-MSMS analysis of BTK using compound as a variable modification search parameter. Inhibition potency of tirabrutinib, ibrutinib, acalabrutinib, and spebrutinib against BTK and related kinases was studied in a dose-dependent manner either after a fixed incubation time (as used in conventional IC₅₀ studies) or following a time course where inactivation kinetics were measured.ResultsTirabrutinib irreversibly and covalently binds to BTK Cys-481. The inactivation efficiency k_inact/K_i was measured and used to calculate selectivity among different kinases for each of the four inhibitors studied. Tirabrutinib showed a k_inact/K_i value of 2.4 ± 0.6 × 10⁴ M⁻¹ s⁻¹ for BTK with selectivity against important off-targets.ConclusionsFor the BTK inhibitors tested in this study, analysis of the inactivation kinetics yielded a more accurate measurement of potency and selectivity than conventional single-time point inhibition measurements. Subtle but clear differences were identified between clinically tested BTK inhibitors which may translate into differentiated clinical efficacy and safety.General significanceThis is the first study that offers a detailed side-by-side comparison of four clinically-relevant BTK inhibitors with respect to their inactivation of BTK and related kinases.     

Potassium-p-nitrophenyl phosphate interactions with (Na+ + K+)-ATPase their relevance to phosphatase activity

The K⁺-dependent p-nitrophenylphosphatase activity catalyzed by purified (Na⁺ + K⁺)-ATPase from pig kidney shows substrate inhibition (K_i about 9.5 mM at 2.1 mM Mg²⁺). Potassium antagonizes and sodium favours this inhibition. In addition, K⁺ reduces the apparent affinity for substrate activation, whereas p-nitrophenyl phosphate reduces the apparent affinity for K⁺ activation. In the absence of Mg²⁺, p-nitrophenyl phosphate, as well as ATP, accelerates the release of Rb⁺ from the Rb⁺ occluded unphosphorylated enzyme. With no Mg²⁺ and with 0.5 mM KCl, trypsin inactivation of (Na⁺ + K⁺)-ATPase as a function of time follows a single exponential but is transformed into a double exponential when 1 mM ATP or 5 mM p-nitrophenyl phosphate are also present. In the presence of 3 mM MgCl₂, 5 mM p-nitrophenyl phosphate and without KCl the trypsin inactivation pattern is that described for the E₁ enzyme form; the addition of 10 mM KCl changes the pattern which, after about 6 min delay, follows a single exponential. These results suggest that (i) the shifting of the enzyme toward the E₁ state is the basis for substrate inhibition of the p-nitrophenulphosphatase acitivy of (Na⁺ + K⁺)-ATPase, and (ii) the substrate site during phosphatase activity is distinct from the low-affinity ATP site.     

Improved Inhibitor Screening Experiments by Comparative Analysis of Simulated Enzyme Progress Curves

A difficulty associated with high throughput screening for enzyme inhibitors is to establish reaction conditions that maximize the sensitivity and resolution of the assay. Deduction of information from end-point assays at single concentrations requires a detailed understanding of the time progress of the enzymatic reaction, an essential but often difficult process to model. A tool to simulate the time progress of enzyme catalyzed reactions and allows adjustment of reactant concentrations and parameters (initial concentrations, K _m, k _cat, K _i values, enzyme half-life, product•enzyme dissociation constant, and the rate constant for the reversed reaction) has been developed. This tool provides comparison of the progress of uninhibited versus inhibited reactions for common inhibitory mechanisms, and guides the tuning of reaction conditions. Possible applications include: analysis of substrate turnover, identification of the point of maximum difference in product concentration (Δ_max[P]) between inhibited and uninhibited reactions, determination of an optimal observation window unbiased for inhibitor mechanisms or potency, and interpretation of observed inhibition in terms of true inhibition. An important observation that can be utilized to improve assay signal strength and resolution is that Δ_max[P] occurs at a high degree of substrate consumption (commonly >75%) and that observation close to this point does not adversely affect observed inhibition or IC₅₀ values.     

β-Glucosidase 2 (GBA2) Activity and Imino Sugar Pharmacology

β-Glucosidase 2 (GBA2) is an enzyme that cleaves the membrane lipid glucosylceramide into glucose and ceramide. The GBA2 gene is mutated in genetic neurological diseases (hereditary spastic paraplegia and cerebellar ataxia). Pharmacologically, GBA2 is reversibly inhibited by alkylated imino sugars that are in clinical use or are being developed for this purpose. We have addressed the ambiguity surrounding one of the defining characteristics of GBA2, which is its sensitivity to inhibition by conduritol B epoxide (CBE). We found that CBE inhibited GBA2, in vitro and in live cells, in a time-dependent fashion, which is typical for mechanism-based enzyme inactivators. Compared with the well characterized impact of CBE on the lysosomal glucosylceramide-degrading enzyme (glucocerebrosidase, GBA), CBE inactivated GBA2 less efficiently, due to a lower affinity for this enzyme (higher K_I) and a lower rate of enzyme inactivation (k_inact). In contrast to CBE, N-butyldeoxygalactonojirimycin exclusively inhibited GBA2. Accordingly, we propose to redefine GBA2 activity as the β-glucosidase that is sensitive to inhibition by N-butyldeoxygalactonojirimycin. Revised as such, GBA2 activity 1) was optimal at pH 5.5–6.0; 2) accounted for a much higher proportion of detergent-independent membrane-associated β-glucosidase activity; 3) was more variable among mouse tissues and neuroblastoma and monocyte cell lines; and 4) was more sensitive to inhibition by N-butyldeoxynojirimycin (miglustat, Zavesca®), in comparison with earlier studies. Our evaluation of GBA2 makes it possible to assess its activity more accurately, which will be helpful in analyzing its physiological roles and involvement in disease and in the pharmacological profiling of monosaccharide mimetics.     

The interaction of bromopyruvate with the active site of 2-keto-3-deoxygluconate-6-phosphate aldolase ofPseudomonas saccharophila: Kinetics and stereochemistry

The interaction of bromopyruvate with the active site of 2-keto-3-deoxygluconate-6-P aldolase ofPseudomonas saccharophila was investigated. The reagent inactivates the enzyme, exhibiting saturation kinetics and competition with pyruvate. The minimal inactivation half-time was 6 min, equivalent to a first-order rate constant of 0.115 min^?1. The concentration of bromopyruvate giving the half-maximal inactivation rateK _inact was 50 mM. TheK _s value of pyruvate as a competitive inhibitor was 0.85 mM. The enzyme asymmetrically detritiates (3RS)-[3? ³H ₂ ]bromopyruvate, forming, in water, (3S)-[3-³H,H]bromopyruvate. This stereochemistry is also exhibited by 2-keto-6-deoxygalactonate-6-P aldolase isolated from the same organism as well as the 2-keto-3-deoxygluconate-5-P aldolase ofP. putida. Over a range of [3-³H]bromopyruvate concentrations affecting the inactivation rate, the ratio of nanomoles reagent catalytically turned over per unit of enzyme inactivated remained constant at 14:1, providing evidence that both catalysis and alkylation occur at the same protein site.     

Expression in Haloferax volcanii of 3-Hydroxy-3-Methylglutaryl Coenzyme A Synthase Facilitates Isolation and Characterization of the Active Form of a Key Enzyme Required for Polyisoprenoid Cell Membrane Biosynthesis in Halophilic Archaea

Enzymes of the isoprenoid biosynthetic pathway in halophilic archaea remain poorly characterized, and parts of the pathway remain cryptic. This situation may be explained, in part, by the difficulty of expressing active, functional recombinant forms of these enzymes. The use of newly available expression plasmids and hosts has allowed the expression and isolation of catalytically active Haloferax volcanii 3-hydroxy-3-methylglutaryl coenzyme A (CoA) synthase (EC 2.3.310). This accomplishment has permitted studies that represent, to the best of our knowledge, the first characterization of an archaeal hydroxymethylglutaryl CoA synthase. Kinetic characterization indicates that, under optimal assay conditions, which include 4 M KCl, the enzyme exhibits catalytic efficiency and substrate saturation at metabolite levels comparable to those reported for the enzyme from nonhalophilic organisms. This enzyme is unique in that it is the first hydroxymethylglutaryl CoA synthase that is insensitive to feedback substrate inhibition by acetoacetyl-CoA. The enzyme supports reaction catalysis in the presence of various organic solvents. Haloferax 3-hydroxy-3-methylglutaryl CoA synthase is sensitive to inactivation by hymeglusin, a specific inhibitor known to affect prokaryotic and eukaryotic forms of the enzyme, with experimentally determined K_i and k_inact values of 570 ± 120 nM and 17 ± 3 min⁻¹, respectively. In in vivo experiments, hymeglusin blocks the propagation of H. volcanii cells, indicating the critical role that the mevalonate pathway plays in isoprenoid biosynthesis by these archaea     

A graphical method for determining inhibition constants

A new simple graphical method is described for the determination of inhibition type and inhibition constants of an enzyme reaction without any replot. The method consists of plotting experimental data as (V–v)/v versus the inhibitor concentration at two or more concentrations of substrate, where V and v represent the maximal velocity and the velocity in the absence and presence of inhibitor with given concentrations of the substrate, respectively. Competitive inhibition gives straight lines that converge on the abscissa at a point where [I]?=??K_i. Uncompetitive inhibition gives parallel lines with the slope of 1/K’_i. For mixed type inhibition, the intersection in the plot is given by [I]?=??K_i and (V–v)/v?=??K_i/K’_i in the third quadrant, and in the special case where K_i?=?K’_i (noncompetitive inhibition) the intersections occur at the point where [I]?=??K_i and (V–v)/v?=??1. The present method, the “quotient velocity plot,” provides a simple way of determining the inhibition constants of all types of inhibitors.     

Kinetic Analysis of the Noncompetitive Inhibition of the Lignin-Peroxidase-catalyzed Reaction by Oxalic Acid

Oxalic acid was found to inhibit noncompetitively the Cα-Cβ bond cleavage of veratrylglycerol catalyzed by a lignin peroxidase (LiP) isozyme of the white-rot fungus P. chrysosporium. With greater amounts of oxalic acid in the LiP system, the substrate was not converted to veratraldehyde but was almost all recovered. Oxalic acid was shown to be decomposed to CO₂ during the enzymatic reaction. The results clearly indicate that oxalic acid reduced the cation radical intermediate formed in the reaction back to the substrate to block the production of veratraldehyde. A novel equation has been derived to explain the mechanism for this unique non-competitive inhibition that is different from the classical noncompetitive one. The inhibition constant K_i obtained here, which is different from the classical inhibition constant K_i, is defined as the ratio of the rate constant (k_p) for product formation to the rate constant (k_i) for the reduction of the cation radical to the substrate.     

α2 High molecular mass cysteine proteinase inhibitor: HMrα2-CPI: An inhibitor of human liver cathepsin H as probed by kinetic study

HMrα₂CPI was found to be an inhibitor of human liver cathepsin H by the measurement of the dissociation constant (K_i), the association rate constant (k₁) and the dissociation rate constant (k_?1) between the enzyme and the inhibitor. These data suggest that this protein-proteinase inhibitor can play a physiological role in the regulation of free cathepsin H.     

Thiamin transport in Escherichia coli: the mechanism of inhibition by the sulfhydryl-specific modifier N-ethylmaleimide

Active transport of thiamin (vitamin B₁) into Escherichia coli occurs through a member of the superfamily of transporters known as ATP-binding cassette (ABC) transporters. Although it was demonstrated that the sulfhydryl-specific modifier N-ethylmaleimide (NEM) inhibited thiamin transport, the exact mechanism of this inhibition is unknown. Therefore, we have carried out a kinetic analysis of thiamin transport to determine the mechanism of inhibition by NEM. Thiamin transport in vivo exhibits Michaelis-Menten kinetics with K_M=15 nM and V_max=46 U mg⁻¹. Treatment of intact E. coli KG33 with saturating NEM exhibited apparent noncompetitive inhibition, decreasing V_max by approximately 50% without effecting K_M or the apparent first-order rate constant (k_obsd). Apparent noncompetitive inhibition is consistent with an irreversible covalent modification of a cysteine(s) that is critical for the transport process. A primary amino acid analysis of the subunits of the thiamin permease combined with our kinetic analysis suggests that inhibition of thiamin transport by NEM is different from other ABC transporters and occurs at the level of protein-protein interactions between the membrane-bound carrier protein and the ATPase subunit.     

Cyclobranol: a substrate for C25-methyl sterol side chains and potent mechanism-based inactivator of plant sterol methyltransferase

Cyclobranol 8A, an analog of the cycloartenol substrate 1A for the plant sterol C24-methyltransferase (SMT), was shown to be an acceptor of the soybean SMT1 as well as an inhibitor of enzyme action. The K_m and k_cat for 8A was 37 μM and 0.006 min⁻¹, respectively. The enzyme-generated product was identified by MS and ¹H NMR to be a C24, C25-doubly alkylated Δ²⁴⁽²⁸⁾-olefin 10A. Inhibitor treatment was concentration and time-dependent affording an apparent K_i of 25 μM, a maximum rate of inactivation of 0.15 min⁻¹ and a partition ratio (k_cat/k_inact) calculated to be 0.04.     

Affinity Labeling of Oxaloacetate Decarboxylase by Novel Dichlorotriazine Linked α-Ketoacids

The 4-aminophenyloxanilic acid and β-mercaptopyruvic acid linked to the reactive diclorotriazine ring, were studied as active site-direct affinity labels towards oxaloacetate decarboxylase (EC 4.1.1.3, OXAD). Oxaloacetate decarboxylase when incubated with 4-aminophenyloxanilic-diclorotriazine (APOD) or β-mercaptopyruvic-diclorotriazine (MPD) at pH 7.0 and 25°C shows a time-dependent and concentration-dependent loss of enzyme activity. The inhibition was irreversible and activity cannot be recovered either by extensive dialysis or gel-filtration chromatography. The enzyme inactivation following the Kitz & Wilson kinetics for time-dependent irreversible inhibition. The observed rate of enzyme inactivation (k _obs) exhibits a non-linear dependence on APOD or MPD concentration with maximum rate of inactivation (k ₃) of 0.013 min^?1 and 0.0046 min^?1 and K _D equal to 20.3 and 156 μM respectively. The inactivation of oxaloacetate decarboxylase by APOD and MPD is competitively inhibited by OXAD substrate and inhibitors, such as oxaloacetate, ADP and oxalic acid whereas Mn⁺² enhances the rate of inactivation. The rate of inactivation of OXAD by APOD shows a pH dependence with an inflection point at 6.8, indicating a possible histidine derivatization by the label. These results show that APOD and MPD demonstrate the characteristics of an active-site probe towards the oxaloacetate binding site of oxaloacetate decarboxylase.     

Catechol-based inhibitors of bacterial urease

Targeted covalent inhibitors of urease were developed on the basis of the catechol structure. Forty amide and ester derivatives of 3,4-dihydroxyphenylacetic acid, caffeic acid, ferulic acid and gallic acid were obtained and screened against Sporosarcinia pasteurii urease. The most active compound, namely propargyl ester of 3,4-dihydroxyphenylacetic acid exhibited IC₅₀?=?518?nM and$k_{\mathit{inact}}/K_i$?=?1379?M^?1?s^?1. Inhibitory activity of this compound was better and toxicity lower than those obtained for the starting compound – catechol. The molecular modelling studies revealed a mode of binding consistent with structure-activity relationships.     

A comparative study of activity and apparent inhibition of fungal β‐glucosidases

β‐Glucosidases (BGs) from Aspergillus fumigates, Aspergillus niger, Aspergillus oryzae, Chaetomium globosum, Emericella nidulans, Magnaporthe grisea, Neurospora crassa, and Penicillium brasilianum were purified to homogeneity, and analyzed by isothermal titration calorimetry with respect to their hydrolytic activity and its sensitivity to glucose (product) using cellobiose as substrate. Global non‐linear regression of several reactions, with or without added glucose, to a product inhibition equation enabled the concurrent derivation of the kinetic parameters k_cat, K_m, and the apparent product inhibition constant _appK_i for each of the enzymes. A more simple fit is not advisable to use as the determined _appK_i are in the same range as their K_m for some of the tested BGs and produced glucose would in these cases interfere. The highest value for k_cat was determined for A. fumigatus (768 s^?1) and the lowest was a factor 9 less. K_m varied by a factor of 3 with the lowest value determined for C. globosum (0.95 mM). The measured _appK_i varied a factor of 15; the hydrolytic activity of N. crassa being the most resistant to glucose with an apparent product inhibition constant of 10.1 mM. Determination of _appK_i using cellobiose as substrate is important as it reflects to what extent the different BGs are hydrolytically active under industrial conditions where natural substrates are hydrolyzed and the final glucose concentrations are high. Biotechnol. Bioeng. 2010;107: 943–952. © 2010 Wiley Periodicals, Inc.