Substrate variability as a factor in enzyme inhibitor design: inhibition of ovine brain glutamine synthetase by alpha- and gamma-substituted phosphinothricins

Ovine brain glutamine synthetase (GS) utilizes various substituted glutamic acids as substrates. We have used this information to design alpha- and gamma-substituted analogues of phosphinothricin [L-2-amino-4-(hydroxymethylphosphinyl)butanoic acid], a naturally occurring inhibitor of GS. These compounds display competitive inhibition of GS, and a correlation between the inhibitor Ki values and the Km/Vmax values of the analogously substituted glutamates supports the hypothesis that the phosphinothricins participate in transition-state analogue inhibition of GS. At concentrations greater than Ki these inhibitors caused biphasic time-dependent loss of enzyme activity, with initial pseudo-first-order behavior; k'inact parameters were determined for several compounds and were similar to the 2.1 X 10(-2)s-1 value measured for PPT. Dilution after GS inactivation caused a non-first-order recovery of activity. Reactivation kinetics were insensitive to inhibitor and ADP concentrations over wide ranges, although very high postdilution concentrations of inhibitor suppressed reactivation. The burst activity level, beta, as well as the concentration of inhibitor required to suppress reactivation to this level, mu, expressed as a multiple of the Ki value, was characteristic for each compound in the phosphinothricin series. Increasing substitution of the phosphinothricin parent structure caused an increase in Ki values as well as in the inactivation/reactivation parameters. The kinetic behavior of these inhibitors is consistent with a mechanistic scheme involving initial phosphorylation and rapid partial inhibitor dissociation, followed by slow release of remaining bound inhibitor.     

Inhibition of tryptophanyl-tRNA synthetase by modifying ATP analogs

The interaction between tryptophanyl-tRNA synthetase (EC 6.1.1.2) from beef pancreas and the ATP analogs containing alkylating or phosphorylating groups in the polyphosphate moiety of ATP was studied as an approach to investigate the structure of the enzyme active center. Some of the compounds under study were shown to irreversibly inhibit the enzyme activity; the presence of ATP in the most cases protects the enzyme against inactivation. The kinetic constants Ki and k2 of interaction of the irreversible inhibitors with the enzyme were determined. It was found that the Ki values for a number of irreversible competitive inhibitors are by 1-2 orders of magnitude less than the Km value for ATP; the k2 values were found equal to 0.02-0.04 min-1. this suggests that the compounds may be used as affinity reagents, the most efficient ones being adenosine 5'-(beta-chloroethyl phosphate) and mixed AMP-mesithylene carbonic acid anhydride. The absence of a protective effect of ATP in the case of adenosine 5'-(beta-bromoethane phosphonate) and non-competitive type of reversible inhibition inhibition of the enzyme by adenosine 5'-chloromethane phosphonate indicate that the molecule of tryptophanyl-tRNA synthetase contains sites interacting with adenine nucleotides, other than the ATP binding sites of the active center.     

Inhibition of plant glutamine synthetases by substituted phosphinothricins

Glutamine synthetase (GS) utilizes various substituted glutamic acids as substrates. We have used this information to design herbicidal α- and γ-substituted analogs of phosphinothricin (l-2-amino-4-(hydroxymethylphosphinyl)butanoic acid, PPT), a naturally occurring GS inhibitor and a potent herbicide. The substituted phosphinothricins inhibit cytosolic sorghum GS₁ and chloroplastic GS₂ competitively versusl-glutamate, with K_i values in the low micromolar range. At higher concentrations, these inhibitors inactivate glutamine synthetase, while dilution restores activity through enzyme-inhibitor dissociation. Herbicidal phosphinothricins exhibit low K_i values and slow enzyme turnover, as described by reactivation characteristics. Both the GS₁ and GS₂ isoforms of plant glutamine synthetase are similarly inhibited by the phosphinothricins, consistent with the broad-spectrum herbicidal activity observed for PPT itself as well as other active compounds in this series.     

Effect of citreoviridin and isocitreoviridin on beef heart mitochondrial ATPase

Citreoviridin is a toxic metabolite from fungus that has been shown to be an inhibitor of mitochondrial F1-ATPases. Studies of citreoviridin, however, have been compromised by the light-dependent isomerization that it undergoes. The isomerization is a potential source of extensive variability in the studies, if citreoviridin and isocitreoviridin have different kinetic effects and binding properties. Both citreoviridin and isocitreoviridin recently have been purified and have been shown to be stable in the dark. Using the purified isomers, the effects of both citreoviridin and isocitreoviridin on soluble and membrane-bound beef heart mitochondrial F1-ATPase activity were investigated. It was found that citreoviridin was an uncompetitive inhibitor of ATP hydrolysis, and a non-competitive inhibitor of ITP hydrolysis catalyzed by soluble F1-ATPase. Isocitreoviridin had no effect on the hydrolysis of either of the triphosphates catalyzed by soluble F1-ATPase. The inhibition constant, Ki for citreoviridin was determined as 4.5 microM for ATP hydrolysis. The inhibition constants Kii and Kis for ITP hydrolysis were determined as 4.3 and 1.03 microM, respectively. Citreoviridin was an uncompetitive inhibitor of ATP hydrolysis and a noncompetitive inhibitor of ATP synthesis catalyzed by membrane-bound F1-ATPase. The inhibition constant, Ki, for ATP hydrolysis was around 4 microM. For ATP synthesis the inhibition constants were determined as 0.12 and 0.16 microM for Kis and Kii, respectively, when ADP concentration was kept saturating. Isocitreoviridin had no effect on either activity of the membrane-bound enzyme.     

Inhibition of Na,K-ATPase by a new ATP analog, adenosine-5-N'-(2,4-dinitro-5-fluorophenyl)phosphohydrazide.

A new ATP analog, adenosine-5-N'-(2,4-dinitro-5-fluorophenyl) phosphohydrazide (DNPH-AMP), has been synthesized, which is an irreversible inhibitor of Na,K-ATPase. Interaction of the analog with the enzyme in the presence of K+ is described by the scheme: [formula: see text] and corresponding kinetic constants k3 and Ki are found equal to 2.5 min-1 and 1.6 mM. In the presence of Na+ the time course of enzyme inactivation by DNPH-AMP is a biphasic curve in the semilogarithmic plot. The k3 and Ki values calculated for this case according to Fritzsch [Fritzsch (1985) J. Theor. Biol. 117, 397] are equal to 2.45 min-1 and 2.5 mM, respectively. ATP transforms the K(+)-type of Na,K-ATPase inactivation into the one that takes place in the presence of Na+.     

Interaction of substrates with glutamine synthetase after limited proteolysis

Previous studies [Dautry-Varsat, A., Cohen, G. N., & Stadtman, E.R. (1979) J. Biol. Chem. 254, 3124-3128; Lei, M., Aebi, U., Heidner, E. G., & Eisenberg, D. (1979) J. Biol. Chem. 254, 3129-3134] have shown that Escherichia coli glutamine synthetase (GS) can be cleaved by proteases to form a limited digestion species called nicked glutamine synthetase (GS). The present study gives the amino acid sequence of the protease-sensitive region of glutamine synthetase. The present study also shows that GS is enzymatically active, but this activity is low compared to the activity of GS. The apparent Michaelis constant value for glutamate was 90 mM for GS as compared to 3 mM for GS, while the Michaelis constant values for ATP were similar for GS and GS*. The dissociation constant values for ATP, as determined by intrinsic fluorescence measurements, were similar for GS and GS*. Glutamate decreased the dissociation constant value of ATP for GS because of synergism between the two binding sites; glutamate did not decrease the dissociation constant value of ATP for GS*. The glutamate analogue methionine sulfoximine bound very tightly to GS and inactivated the enzyme in the presence of ATP. Methionine sulfoximine did not appear to bind to GS* and did not inactivate GS* in the presence of ATP. The ATP analogue 5'-[p-(fluorosulfonyl)benzoyl]adenosine bound to GS and inactivated the enzyme by forming a covalent bond with it. Glutamate accelerated this inactivation because of the synergism between the ATP and glutamate binding sites of GS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition kinetics of green crab (Scylla serrata) alkaline phosphatase activity by dithiothreitol or 2-mercaptoethanol

Green crab (Scylla serrata) alkaline phosphatase (EC 3.1.3.1) is a metalloenzyme which catalyzes the nonspecific hydrolysis of phosphate monoesters. Some pollutants in seawater affect the enzyme activity causing loss of the biological function of the enzyme, which affects the exuviating crab-shell and threatens the survival of the animal. The present paper studies the effects of thiohydroxyal compounds on the activity of green crab alkaline phosphatase. The results show that thiohydroxyal compounds can lead to reversible inhibition. The equilibrium constants have been determined for dithiothreitol (DTT) and mercaptoethanol (ME) binding with the enzyme and/or the enzyme-substrate complexes. The results show that both DTT and ME are non-competitive inhibitors. The kinetics of enzyme inactivation by ME at low concentrations has been studied using the kinetic method of the substrate reaction. The results suggest that at pH 10.0, the action of ME on green crab ALP is first quick equilibrium binding and then slow inactivation. The microscopic rate constants were determined for inactivation and reactivation. The rate constant of the forward inactivation (k(+0)) is much larger than that of the reverse reactivation (k(-0)). Therefore, when the ME concentration is sufficiently large, the enzyme is completely inactivated.     

Inactivation of delta 5-3-oxo steroid isomerase with active-site-directed acetylenic steroids.

Several steroid analogues containing conjugated acetylenic ketone groups as part of a seco-ring structure or as substituents on the intact steroid system are irreversible inhibitors of delta 5-3-oxo steroid isomerase (EC 5.3.3.1) from Pseudomonas testosteroni. Thus 10 beta-(1-oxoprop-2-ynyl)oestr-4-ene-3,17-dione (I), 5,10-seco-oestr-4-yne-3,10,17-trione (II), 17 beta-hydroxy-5,10-seco-oestr-4-yne-3,10-dione (III) and 17 beta-(1-oxoprop-2-ynyl)androst-4-en-3-one (IV) irreversibly inactivate isomerase in a time-dependent manner. In all cases saturation kinetics are observed. Protection against inactivation is afforded by the powerful competitive inhibitor 19-nortestosterone. The inhibition constants (Ki) for 19-nortestosterone obtained from such experiments are in good agreement with those determined from conventional competitive-inhibition studies of enzyme activity. These compounds thus appear to be active-site directed. In every case the inactivated enzyme could be dialysed without return of activity, indicating that a stable covalent bond probably had formed between the steroid and enzyme. Compound (I) is a very potent inhibitor of isomerase [Ki = 66.0 microM and k+2 = 12.5 x 10(-3) s-1 (where Ki is the dissociation constant of the reversible enzyme-inhibitor complex and k+2 is the rate constant for the inactivation reaction of the enzyme-inhibitor complex)] giving half-lives of inactivation of 30-45 s at saturation. It is argued that the basic-amino-acid residue that abstracts the intramolecularly transferred 4 beta-proton in the reaction mechanism could form a Michael-addition product with compound (I). In contrast, although compound (IV) has a lower inhibition constant (Ki = 14.5 microM), it is a relatively poor alkylating agent (k+2 = 0.13 x 10(-3) s-1). If the conjugated acetylenic ketone groups are replaced by alpha-hydroxyacetylene groups, the resultant analogues of steroids (I)-(IV) are reversible competitive inhibitors with Ki values in the range 27-350 microM. The enzyme binds steroids in the C19 series with functionalized acetylenic substituents at C-17 in preference to steroids in the C18 series bearing similar groups in the ring structure or as C-10 substituents. In the 5,10-seco-steroid series the presence of hydroxy groups at both C-3 and C-17 is deleterious to binding by the enzyme.     

Kinetics of beta-lactamase inhibition by clavulanic acid]

The mechanisms of action of 3 R-factors on beta-lactamases (penicillin amido-beta-lactamhydrolase, EC 3.5.2.6) (TEM-1 pI = 5.4, TEM-2 pI = 5.6 and Pitton's type 2 pI = 7.7) have been kinetically analyzed for clavulanic acid inactivation. Clavulanic acid appears as a competitive and irreversible inhibitor (Kcat inhibitor) reacting in two steps: a, formation of a reversible enzyme . inhibitor complex (characterized by a Ki); b, evolution of the reversible complex into a new derivative (covalent, stable and inactive) by monomolecular kinetics characterized by a k6 (or Kcat) related to half-life. The kinetic constants are: TEM-1: Ki = 0.8 micrometer, k6 = 0.027 s-1; TEM-2: Ki = 0.7 micrometer, k6 = 0.03 s-1; type 2: Ki = 0.6 micrometer, k6 = 0.046 s-1. These results justify the 'progressive irreversible' character of the inhibition generally described.     

A specific inhibitor of the ubiquitin activating enzyme: synthesis and characterization of adenosyl-phospho-ubiquitinol, a nonhydrolyzable ubiquitin adenylate analogue

A nonhydrolyzable analogue of ubiquitin adenylate has been synthesized for use as a specific inhibitor of the ubiquitination of proteins. Ubiquitin adenylate is a tightly bound intermediate formed by the ubiquitin activating enzyme. The inhibitor adenosyl-phospho-ubiquitinol (APU) is the phosphodiester of adenosine and the C-terminal alcohol derived from ubiquitin. APU is isosteric with the normal reaction intermediate, the mixed anhydride of ubiquitin and AMP, but results from the replacement of the carbonyl oxygen of Gly76 with a methylene group. This stable analogue would be expected to bind to both ubiquitin and adenosine subsites and result in a tightly bound competitive inhibitor of ubiquitin activation. APU inhibits the ATP-PPi exchange reaction catalyzed by the purified ubiquitin activating enzyme in a manner competitive with ATP (Ki = 50 nM) and noncompetitive with ubiquitin (Ki = 35 nM). AMP has no effect on the inhibition, confirming that the inhibitor binds to the free form of the enzyme and not the thiol ester form. This inhibition constant is 10-fold lower than the dissociation constants for each substrate and 30-1000-fold lower than the respective Km values for ubiquitin and ATP. APU also effectively inhibits conjugation of ubiquitin to endogenous proteins catalyzed by reticulocyte fraction II with an apparent Ki of 0.75 microM. This weaker inhibition is consistent with the fact that activation of ubiquitin is not rate limiting in the conjugation reactions catalyzed by fraction II. APU is similarly effective as an inhibitor of the ubiquitin-dependent proteolysis of beta-lactoglobulin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of ATP hydrolysis catalyzed by isolated TF1 and reconstituted TF0F1 ATPase

The rate of ATP hydrolysis catalyzed by isolated TF1 and reconstituted TF0F1 was measured as a function of the ATP concentration in the presence of inhibitors [ADP, Pi and 3'-O-(1-naphthoyl)ATP]. ATP hydrolysis can be described by Michaelis-Menten kinetics with Km(TF1) = 390 microM and Km (TF0F1) = 180 microM. The inhibition constants are for ADP Ki(TF1) = 20 microM and Ki(TF0F1) = 100 microM, for 3'-O-(1-naphthoyl)ATP Ki(TF1) = 150 microM and Ki(TF0F1) = 3 microM, and for Pi Ki(TF1) = 60 mM. From these results it is concluded that upon binding of TF0 to TF1 the mechanism of ATP hydrolysis catalyzed by TF1 is not changed qualitatively; however, the kinetic constants differ quantitatively.     

Role of charge and hydrophobic effects in reactions of peptide substrates and inhibitors with thrombin

A substrate and inhibitor analysis of the thrombin interaction with synthetic peptide substrates and inhibitors of differing hydrophobicity and volume of the side amino acid residue, localized in the sub-centers thrombin S2 and S3 were carried out. The kinetic parameters of individual stages of the enzymatic reaction process (K(S), k2, k3) were estimated. It is shown that the efficiency of acylation and deacylation stages of the enzymatic reaction decreases with increasing hydrophobicity of the substituent in P2 as well as P3, at the same time the affinity of selected peptides toward enzyme is steadily increasing. With the aim to evaluate the hydrophobicity of compounds a LogP value was calculated and was made an attempt to compare them with the correspondent Ki values. Comparative kinetic analysis of Z-Arg-OMe and its uncharged analogue Z-Cit-OMe has shown the absence of uncharged analog hydrolysis, however, the mentioned citrulline derivate inhibits the hydrolysis of the charged analogue. These findings confirm the important role of hydrophobic moiety in the structure of thrombin inhibitors in preferential binding mode and inhibition of thrombin active side.     

Evidence for the existence of a sulfhydryl group in the adenylate cyclase active site

6-Cloro-9-beta-d-ribofuranosylpurine 5'-triphosphate (CIRTP) and 6-mercapto-9-beta-d-ribofuranosylpurine 5'-triphosphate (SRTP) irreversibly inhibit adenylate cyclase from rat brain. Adenosine 5'-[beta, gamma -imido] triphosphate protects the enzyme against inactivation by CIRTP and SRTP and acts as a competitive inhibitor with respect to ATP with the Ki value 2 X 10(-4) M. Study of the pH-dependence of the rate of the enzyme inactivation by CIRTP showed that pK for the group modified by this compound is equal to 7.45. Inactivation is first order with respect to the enzyme; the saturation effect is observed at the increased concentration of CIRTP. The k2 and KI values for irreversible inhibition of brain adenylate cyclase by CIRTP were 0.25 min-1 and 1.9 X 10(-4) M, respectively. Adenylate cyclase inhibition by SRTP is also time-dependent. Partial protection against the enzyme inactivation was observed. Dithiothreitol restores the activity of SRTP-inactivated adenylate cyclase. The results obtained indicate the presence of an -SH group in the purine amino group binding area of the enzyme active site.     

Effects of temperature and ethanol on agonist and antagonist binding to rat heart muscarinic receptors in the absence and presence of GTP.

2 alpha-Cyanoprogesterone (I) and 2-hydroxymethyleneprogesterone (II) were synthesized and screened as irreversible active-site-directed inhibitors of the delta 5-3-oxosteroid isomerase (EC 5.3.3.1) from Pseudomonas testosteroni. Both compounds were found to inhibit the purified bacterial enzyme in a time-dependent manner. In either case the inactivated enzyme could be dialysed without return of activity, indicating that a stable covalent bond had formed between the inhibitor and the enzyme. Inactivation mediated by compounds (I) and (II) followed pseudo-first-order kinetics, and at higher inhibitor concentrations saturation was observed. The competitive inhibitor 17 beta-oestradiol offered protection against the inactivation mediated by both compounds, and initial-rate studies indicated that compounds (I) and (II) can also act as competitive inhibitors yielding Ki values identical with those generated during inactivation experiments. 2 alpha-Cyanoprogesterone (I) and 2-hydroxymethyleneprogesterone (II) thus appear to be active-site-directed. To compare the reactivity of these 2-substituted progesterones with other irreversible inhibitors of the isomerase, 3 beta-spiro-oxiranyl-5 alpha-pregnan-20 beta-ol (III) was synthesized as the C21 analogue of 3 beta-spiro-oxiranyl-5 alpha-androstan-17 beta-ol, which is a potent inactivator of the isomerase [Pollack, Kayser & Bevins (1979) Biochem. Biophys. Res. Commun. 91, 783-790]. Comparison of the bimolecular rate constants for inactivation (k+3/Ki) mediated by compounds (I)-(III) indicated the following order of reactivity: (III) greater than (II) greater than (I). 2-Mercaptoethanol offers complete protection against the inactivation of the isomerase mediated by 2 alpha-cyanoprogesterone (I). Under the conditions of inactivation compound (I) appears to be completely stable, and no evidence could be obtained for enolate ion formation in the presence or absence of enzyme. It is suggested that cyanoprogesterone inactivates the isomerase after direct nucleophilic attack at the electropositive 2-position, and that tautomerization plays no role in the inactivation event. By contrast, 2-mercaptoethanol offers no protection against the inactivation mediated by 2-hydroxymethyleneprogesterone, and under the conditions of inactivation this compound appears to exist in the semi-enolized form.     

Molecular recognition by acetylcholinesterase at the peripheral anionic site: structure-activity relationships for inhibitions by aryl carbamates

Substituted phenyl-N-butyl carbamates (1-9) are potent irreversible inhibitors of Electrophorus electricus acetylcholinesterase. Carbamates 1-9 act as the peripheral anionic site-directed irreversible inhibitors of acetylcholinesterase by the stop-time assay in the presence of a competitive inhibitor, edrophonium. Linear relationships between the logarithms of the dissociation constant of the enzyme inhibitor adduct (Ki), the inactivation constant of the enzyme-inhibitor adduct (k2), and the bimolecular inhibition constant (k(i)) for the inhibition of Electrophorus electricus acetylcholinesterase by carbamates 1-9 and the Hammett substituent constant (sigma), are observed, and the reaction constants (ps) are -1.36, 0.35 and -1.01, respectively. Therefore, the above reaction may form a positive charged enzyme-inhibitor intermediate at the peripheral anionic site of the enzyme and may follow the irreversible inactivation by a conformational change of the enzyme.     

Two-step binding mechanism for HIV protease inhibitors.

Rate constants for binding of five inhibitors of human immunodeficiency virus (HIV) protease were determined by stopped-flow spectrofluorometry. The two isomers of quinoline-2-carbonyl-Asn-Phe psi-[CH(OH)CH2N]Pro-O-t-Bu (R diastereomer = 1R; S diastereomer = 1S) quenched the protein fluorescence of HIV protease and thus provided a spectrofluorometric method to determine their binding rate constants. The dissociation rate constants for acetyl-Thr-Ile-Leu psi(CH2NH)Leu-Gln-Arg-NH2 (2), (carbobenzyloxy)-Phe psi[CH(OH)CH2N]Pro-O-t-Bu (3), and pepstatin were determined by trapping free enzyme with 1R as 2, 3, and pepstatin dissociated from the respective enzyme.inhibitor complex. Association rate constants of 1R, 2, and pepstatin were calculated from the time-dependent inhibition of protease-catalyzed hydrolysis of the fluorescent substrate (2-aminobenzoyl)-Thr-Ile-Nle-Phe(NO2)-Gln-Arg-NH2 (4). The kinetic data for binding of 1S to the protease fit a two-step mechanism. Kd values for these inhibitors were calculated from the rate constants for binding and were similar to the respective steady-state Ki values.     

Competitive binding of ATP and the fluorescent substrate analogue 2',3'-O-(2,4,6-trinitrophenylcyclohexadienylidine) adenosine 5'-triphosphate to the gastric H+,K+-ATPase: evidence for two classes of nucleotide sites

ATP and the fluorescent substrate analogue TNP-ATP bind competitively to the gastric H,-K-ATPase. Substrate and product completely reverse the fluorescence enhancement caused by TNP-ATP binding to the enzyme. The fluorophore is displaced monophasically from apoenzyme. However, ATP displaces TNP-ATP from the Mg2+-quenched state in two steps of equal amplitude. The midpoints of the titrations differ by more than 2 orders of magnitude. The estimated substrate constants are in reasonable agreement with published Michaelis constants. TNP-ATP is not a substrate for the H,K-ATPase. The fluorophore prevents phosphorylation by ATP and competitively inhibits the K+-stimulated pNPPase and ATPase activities of the enzyme. Ki is approximately the same for both hydrolytic activities and consistent with the Kd of TNP-ATP measured directly. Km for pNPP is 1.48 +/- 0.15 mM. Two Michaelis constants are required to fit the ATPase data: Km1 = 0.10 +/- 0.01 mM and Km2 = 0.26 +/- 0.05 mM.     

The effects of exchange-inert metal-nucleotide complexes on the kinetics of beef heart mitochondrial F1-ATPase

The inhibition of beef heart mitochondrial F1 by exchange-inert metal-nucleotide complexes was examined. Mono- and bidentate Cr(NH3)4ATP were found to be mixed noncompetitive inhibitors of F1-catalyzed ATP hydrolysis (values of Ki = 0.5 and 0.1 mM; values of alpha = 0.2 and 24, respectively). Rh(H2O)nATP was also found to be a mixed noncompetitive inhibitor of F1-catalyzed ATP hydrolysis (Ki = 0.3 mM, alpha = 0.7). These compounds were used in a series of dual inhibition experiments, along with mono- and bidentate CrATP and Co(NH3)4ATP. All the exchange-inert metal-nucleotides examined were found to be mutually exclusive inhibitors of F1, indicating that they all bind to the same site(s). It is postulated that the pKa of the metal-coordinated ligands is related to the potency of inhibition by these compounds. It appears probable that the exchange-inert nucleotide complexes are binding to site(s) in addition to the catalytic site(s) of F1.     

Half-time analysis of the kinetics of irreversible enzyme inhibition by an unstable site-specific reagent

The half-time method for the determination of Michaelis parameters from enzyme progress-curve data (Wharton, C.W. and Szawelski, R.J. (1982) Biochem. J. 203, 351-360) has been adapted for analysis of the kinetics of irreversible enzyme inhibition by an unstable site-specific inhibitor. The method is applicable to a model in which a product (R) of the decomposition of the site-specific reagent, retaining the chemical moiety responsible for inhibitor specificity, binds reversibly to the enzyme with dissociation constant Kr: (formula; see text). Half-time plots of simulated enzyme inactivation time-course data are shown to be unbiased, and excellent estimates of the apparent second-order rate constant for inactivation (k +2/Ki) and Kr can be obtained from a series of experiments with varying initial concentrations of inhibitor. Reliable estimates of k +2 and Ki individually are dependent upon the relative magnitudes of the kinetic parameters describing inactivation. The special case, Kr = Ki, is considered in some detail, and the integrated rate equation describing enzyme inactivation shown to be analogous to that for a simple bimolecular reaction between enzyme and an unstable irreversible inhibitor without the formation of a reversible enzyme-inhibitor complex. The half-time method can be directly extended to the kinetics of enzyme inactivation by an unstable mechanism-based (suicide) inhibitor, provided that the inhibitor is not also a substrate for the enzyme.     

Inhibition by excess of free ATP, and free Mg2+ ions of the mitochondrial F1-ATPase moiety from Phycomyces blakesleeanus.

High concentrations of either Mg-ATP complex, free ATP, or free Mg2+ ions were inhibitors of the mitochondrial F1-ATPase moiety from Phycomyces blakesleeanus. Free Mg2+ acts as a linear competitive inhibitor with regard to Mg-ATP hydrolysis with a Ki value of 2.8 mM. The inhibition by free ATP was markedly biphasic and thus simple competitive inhibition alone is not sufficient to explain the inhibitory effect. From these results conclusions were drawn about the binding of the substrate, Mg-ATP complex, to the enzyme.