Quantitative structure-activity relationships for the pre-steady-state inhibition of cholesterol esterase by 4-nitrophenyl-N-substituted carbamates

4-Nitrophenyl-N-substituted carbamates (1-6) are the pseudo-substrate inhibitors of porcine pancreatic cholesterol esterase. Thus, the first step of the inhibition (Ki step) is the formation of the enzyme inhibitor tetrahedral adduct and the second step of the inhibition (kc) is the formation of the carbamyl enzyme. The formation of the enzyme inhibitor tetrahedral adduct is further divided into two steps, the formation of the enzyme-inhibitor complex with the dissociation constant, KS, at the first step and the formation of the enzyme-inhibitor tetrahedral adduct from the complex at the second step. The two-step mechanism for the formation of the enzyme-inhibitor tetrahedral adduct is confirmed by the pre-steady-state kinetics. The results of quantitative structure-activity relationships for the pre-steady-state inhibitions of cholesterol esterase by carbamates 1-6 indicate that values of -logKs and logk2/k-2 are correlated with the Taft substituent constant, sigma*, and the rho* values from these correlations are -0.33 and 0.1, respectively. The negative rho* value for the -logKS-sigma*-correlation indicates that the first step of the two-step formation of the enzyme-inhibitor tetrahedral adduct (KS step) is the formation of the positive enzyme inhibitor complex. The positive rho* value for the logk2/k-2 -sigma*-correlation indicates that the enzyme inhibitor tetrahedral adduct is more negative than the enzyme inhibitor complex. Finally, the two-step mechanism for the formation of the enzyme inhibitor tetrahedral adduct is proposed according to these results. Thus, the partially positive charge is developed at nitrogen of carbamates 1-6 in the enzyme-inhibitor complex probably due to the hydrogen bonding between the lone pair of nitrogen of carbamates 1-6 and the amide hydrogen of the oxyanion hole of the enzyme. The second step of the two-step formation of the enzyme-inhibitor tetrahedral adduct is the nucleophilic attack of the serine of the enzyme to the carbonyl group of carbamates 1-6 in the enzyme-inhibitor complex and develops the negative-charged oxygen in the adduct.     

Quantitative structure-activity relationships for the pre-steady state of Pseudomonas species lipase inhibitions by p-nirophenyl-N-substituted carbamates

The pre-steady states of Pseudomonas species lipase inhibitions by p-nitrophenyl-N-substituted carbamates (1-6) are composed of two steps: (1) formation of the non-covalent enzyme-inhibitor complex (E:I) from the inhibitor and the enzyme and (2) formation of the tetrahedral enzyme-inhibitor adduct (E-I) from the E:I complex. From a stopped-flow apparatus, the dissociation constant for the E:I complex, KS, and the rate constant for formation of the tetrahedral E-I adduct from the E:I complex, k2 are obtained from the non-linear least-squares of curve fittings of first-order rate constant (k(obs)) versus inhibition concentration ([I]) plot against k(obs)=k2+k2[I]/(KS+[I]). Values of pKS, and log k2 are linearly correlated with the sigma* values with the rho* values of -2.0 and 0.36, respectively. Therefore, the E:I complexes are more positive charges than the inhibitors due to the rho* value of -2.0. The tetrahedral E-I adducts on the other hand are more negative charges than the E:I complexes due to the rho* value of 0.36. Formation of the E:I complex from the inhibitor and the enzyme are further divided into two steps: (1) the pre-equilibrium protonation of the inhibitor and (2) formation of the E:I complex from the protonated inhibitor and the enzyme.     

QSAR for phospholipase A2 inhibitions by 1-acyloxy-3-N-n-octylcarbamyl-benzenes

1-Acyloxy-3-N-n-octylcarbamyl-benzenes are potent reversible competitive inhibitors of Naja mocambique mocambique phospholipase A2 with the Ki values from 9.6 to 119 microM. The pKi values are correlated to both Taft substituent constant sigma* and Hansch hydrophobicity constant pi. The pre-steady state inhibition studies indicate that the pK(S) values for the first inhibition step are linearly correlated to sigma* alone with the rho* of -0.09 for this correlation. Thus, the first inhibition step may involve the insertion of the inhibitor to hepta-coordinated Ca2+ ion of the enzyme to form the octa-coordinated Ca2+ ion of the enzyme. The log(k2/k(-2)) values for the second inhibition step are linearly correlated to pi alone, and the psi value for this correlation is 0.13. Therefore, the second step inhibition step may involve the van der Waals' interaction between the acyl group of the inhibitor and Tyr 69 of the enzyme.     

Synthesis and evaluation of a new series of tri-, di-, and mono-N-alkylcarbamylphloroglucinols as conformationally constrained inhibitors of cholesterol esterase

1,3,5-Tri-N-alkylcarbamylphloroglucinols (1-4) are synthesized as conformationally constrained analogs of triacylglycerols (TGs) to probe Jenck's proximity effect in the cholesterol esterase inhibition. For the cholesterol esterase inhibition, inhibitors 1-4 are 220-760-fold more potent than 1,2,3-tri-N-alkylcarbamylglycerols (13-15) that are substrate analogs of TG. Comparison of tridentate inhibitors 1-4, bidentate inhibitors 3,5-di-N-n-alkylcarbamyloxyphenols (5-8) and monodentate inhibitors 5-N-n-alkylcarbamyloxyresorcinols (9-12) indicates that inhibitory potencies are as followed: tridentate inhibitor > bidentate inhibitor > monodentate inhibitor. The log k(i) and pK(i) values of tridentate inhibitors, bidentate inhibitors, and monodentate inhibitors are linearly correlated with the alkyl chain length indicating a common mechanism in each inhibition. Also, positive slopes of these correlations indicate that the longer chain inhibitors bind more tightly to the enzyme than the shorter ones. Molecular dockings of tridentate 1, bidentate 5, and monodentate 9 into the X-ray crystal structure of cholesterol esterase suggest that one carbamyl group in the cis form of the inhibitor binds to the acyl chain-binding site of the enzyme. The second carbamyl groups in the trans forms of inhibitors 1 and 5 bind to the second acyl chain-binding site of the enzyme. The third carbamyl group in the trans form of inhibitor 1 binds to the third acyl chain-binding site of the enzyme. Moreover, the configuration of the inhibitor in the enzyme-inhibitor complex is the (1,3,5)-(cis, trans, trans)-tricarbamate form that mimics the (+gauche, -gauche)-conformation of TG.     

Probing the peripheral anionic site of acetylcholinesterase with quantitative structure activity relationships for inhibition by biphenyl-4-acyoxylate-4'-N-Butylcarbamates

Biphenyl-4-acyoxylate-4'-N-butylcarbamates 1-8 are synthesized from 4,4'-biphenol and are characterized as the pseudosubstrate inhibitors of acetylcholinesterase. In other words, the inhibitors bind to the enzyme and react with the enzyme to form the tetrahedral intermediates for the K(i) steps, and then the tetrahedral intermediates exclude the leaving groups to form a common N-butycarbamyl enzyme intermediate for the k(c) steps. Due to a linear character of the 4,4'-biphenyl moiety, the 4'-N-butylcarbamate moieties of the inhibitors react with the Ser200 residue of the enzyme while the 4-acyoxylate moieties of the inhibitors, on the other hand, should fit in the peripheral anionic site of the enzyme, which is located at the mouth of the deep active site gorge. Thus, carbamates with varied acyl substituents at the 4-position of the biphenyl ring are good candidates for probing the quantitative structure activity relationships for the peripheral anionic site of the enzyme. The fact that the pK(i), log k(c), and log K(i) values are correlated with neither the Taft substituent constant (sigma*) nor the Taft steric constant (E(s)) indicates that the 4-acyoxylate moieties of the inhibitors are too far away from the reaction center. However, the pK(i), log k(c), and log K(i) values are linearly correlated with the Hansch hydrophobicity constant, pi. The intensity constants (psi) for these correlations are 0.16, -0.035, and 0.13, respectively. These results indicate that interactions between the 4-acyoxylate groups of the inhibitors and the peripheral anionic site of the enzyme are mainly hydrophobic ones. The correlation results are slightly improved by using the two-parameter correlations with the Taft substituent steric constant, E(s), and pi. For pK(i), log k(c), and log K(i)-E(s)-pi correlations, the psi values are 0.21, -0.021, and 0.19, respectively; the intensity constants for steric effect (delta) are 0.08, 0.022, and 0.10, respectively. Besides hydrophobic interactions, the two-parameter correlations also suggest that little steric hindrance occurs for the bulkier inhibitors to pass by the peripheral anionic site of the enzyme.     

Ortho effects in quantitative structure-activity relationships for acetylcholinesterase inhibition by aryl carbamates

Ortho-substituted phenyl-N-butyl carbamates (1-9) are characterized as "pseudo-pseudo-substrate" inhibitors of acetylcholinesterase. Since the inhibitors protonate at pH 7.0 buffer solution, the virtual inhibition constants (K'is) of the protonated inhibitors are calculated from the equation, - logK'i = - logKi - logKb. The logarithms of the inhibition constant (Ki), the carbamylation constant (k(c)), and the bimolecular inhibition constant (k(i)) for the enzyme inhibitions by carbamates 1-9 are multiply linearly correlated with the Hammett para-substituent constant (sigma(p)), the Taft-Kutter-Hansch ortho steric constant (E(S)), and the Swan-Lupton ortho polar constant (F). Values of rho, delta, and f for the - logKi-, logk(c)-, and logk(i)-correlations are -0.6, -0.16, 0.7; 0.11, 0.03, -0.3; and - 0.5, - 0.12, 0.4, respectively. The Ki step further divides into two steps: 1) the pre-equilibrium protonation of the inhibitors, Kb step and 2) formation of a negatively charged enzyme-inhibitor Michaelis-Menten complex--virtual inhibition, K'i step. The Ki step has little ortho steric enhancement effect; moreover, the k(c)step is insensitive to the ortho steric effect. The f value of 0.7 for the Ki step indicates that ortho electron-withdrawing substituents of the inhibitors accelerate the inhibition reactions from the ortho polar effect; however, the f value of -0.3 for the k(c)step implies that ortho electron-withdrawing substituents of the inhibitors lessen the inhibition reactions from the ortho polar effect.     

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.     

Linear relationships between the ligand binding energy and the activation energy of time-dependent inhibition of steroid 5alpha-reductase by delta 1-4-azasteroids

The inhibition of steroid 5alpha-reductase (5AR) by Delta(1)-4-azasteroids is characterized by a two-step time-dependent kinetic mechanism where inhibitor combines with enzyme in a fast equilibrium, defined by the inhibition constant K(i), to form an initial reversible enzyme-inhibitor complex, which subsequently undergoes a time-dependent chemical rearrangement, defined by the rate constant k(3), leading to the formation of an apparently irreversible, tight-binding enzyme-inhibitor complex (Tian, G., Mook, R. A., Jr., Moss, M. L., and Frye, S. V. (1995) Biochemistry 34, 13453-13459). A detailed kinetic analysis of this process with a series of Delta(1)-4-azasteroids having different C-17 substituents was performed to understand the relationships between the rate of time-dependent inhibition and the affinity of the time-dependent inhibitors for the enzyme. A linear correlation was observed between ln(1/K(i)), which is proportional to the ligand binding energy for the formation of the enzyme-inhibitor complex, and ln(1/(k(3)/K(i))), which is proportional to the activation energy for the inhibition reaction under the second order reaction condition, which leads to the formation of the irreversible, tight-binding enzyme-inhibitor complex. The coefficient of the correlation was -0.88 +/- 0.07 for type 1 5AR and -1.0 +/- 0.2 for type 2 5AR. In comparison, there was no obvious correlation between ln(1/K(i)) and ln(1/k(3)), which is proportional to the activation energy of the second, time-dependent step of the inhibition reaction. These data are consistent with a model where ligand binding energies provided at C-17 of Delta(1)-4-azasteroids is fully expressed to lower the activation energy of k(3)/K(i) with little perturbation of the energy barrier of the second, time-dependent step.     

Structure-reactivity relationships for the inhibition mechanism at the second alkyl-chain-binding site of cholesterol esterase and lipase.

Alkyl-N-phenyl carbamates (2-8) (see Figure 1), alkyl-N-phenyl thiocarbamates (9-15), 2,2'-biphenyl-2-ol-2'-N-substituted carbamates (16-23), and 2, 2'-biphenyl-2-N-octadecylcarbamate-2'-N-substituted carbamates (24-31) are prepared and evaluated for their inhibition effects on porcine pancreatic cholesterol esterase and Pseudomona species lipase. All inhibitors are characterized as transient or pseudo substrate inhibitors for both enzymes. Both enzymes are not protected from inhibition and further inactivated by carbamates 2-8 and thiocarbamates 9-15 in the presence of trifluoroacetophenone. Therefore, carbamates 2-8 and thiocarbamates 9-15 are exceptions for active site binding inhibitors and are probably the second alkyl-chain binding-site-directed inhibitors for both enzymes. The inhibition data for carbamates 2-8 and thiocarbamates 9-15 are correlated with the steric constant, E(s), and the hydrophobicity constant, pi; however, the inhibition data are not correlated with the Taft substituent constant, sigma. A comparison of the inhibition data for carbamates 2-8 and thiocarbamates 9-15 toward both enzymes indicates that thiocarbamates 9-15 are more potent inhibitors than carbamates 2-8. A comparison of the inhibition data for cholesterol esterase and Pseudomona species lipase by carbamates 2-8 or thiocarbamates 9-15 indicates that cholesterol esterase is more sensitive to the E(s) and pi values than Pseudomona species lipase. The negative slope values for the logarithms of inhibition data for Pseudomona species lipase by carbamates 2-8 and thiocarbamates 9-15 versus E(s) and pi indicate that the second alkyl-chain-binding site of Pseudomona species lipase is huge, hydrophilic, compared to that of cholesterol esterase, and prefers to interact with a bulky, hydrophilic inhibitor rather than a small, hydrophobic one. On the contrary, the second alkyl-chain-binding site of cholesterol esterase prefers to bind to a small, hydrophobic inhibitor. Both enzymes are protected from inhibition by carbamates 16-23 in the presence of trifluoroacetophenone. Therefore, carbamates 16-23 are characterized as the alkyl chain binding site, esteratic site oxyanion active site directed pseudo substrate inhibitors for both enzymes. Both enzyme inhibition data for carbamates 16-22 are well-correlated with sigma alone. The negative rho values for these correlations indicate that the serine residue of both enzymes and carbamates 16-22 forms the tetrahedral species with more positive charges than inhibitors and the enzymes and follow the formation of the carbamyl enzymes with more positive charges than the tetrahedral species. Carbamates 24-31 are also exceptions for active site binding inhibitors and probably the second alkyl chain binding site-directed inhibitors for both enzymes. However, the enzyme inhibition constants for carbamates 24-31 are correlated with values of sigma, E(s), and pi. The negative rho values for these correlations indicate that both enzymes and carbamates 24-31 form the tetrahedral species with more positive charges than inhibitors and the enzymes and follow the formation of the carbamyl enzymes with more positive charges than those tetrahedral species. Therefore, carbamates 24-31 may bind to both the active sites and the second alkyl chain binding site and follow the evacuation of the active sites. A comparison of the rho values for cholesterol esterase and Pseudomona species lipase by carbamates 24-31 indicates that cholesterol esterase is much more sensitive to the sigma values than Pseudomona species lipase. The negative sensitivity values, delta, for the cholesterol esterase inhibitions by carbamates 24-31 indicate that the enzyme prefers to bind to a bulky carbamyl group rather than bind to a small one. The hydrophobicity of carbamates 24-31 does not play a major role in both enzyme inhibitions.     

N-Butyl-N-methyl-4-nitrophenyl carbamate as a specific active site titrator of bile-salt-dependent lipases

The effect of a series of synthetic carbamates on the human (milk or pancreatic) bile-salt-dependent lipase (cholesterol esterase) was examined. N-isopropyl-O-phenyl, N-methyl-O-phenyl, N-butyl-(4-nitrophenyl), N-phenyl-(4-nitrophenyl), N-butyl-N-methyl and N-pentyl-O-phenyl carbamates were inhibitors of the enzyme activity, while O-isopropyl-N-phenyl, O-methyl-N-phenyl, O-benzyl-N-isopropyl and O-cyclohexyl-N-phenyl carbamates were not even recognized by the enzyme. The N-alkyl chain length is essential for the enzyme inhibition and N-butyl-(4-nitrophenyl) or N-pentyl-O-phenyl carbamates are more potent inhibitors than N-methyl-O-phenyl or N-isopropyl carbamates. The inhibition by reactive carbamates fits the criteria for mechanism-based inhibition: the inhibition is first-order with time, shows saturation kinetics with increasing carbamate concentration and leads to an inactive stoichiometric enzyme-inhibitor complex; the enzyme activity can be protected by a competitive inhibitor. Evidence is shown that the enzymatic nucleophilic attack of carbamates is directed at the carbonyl carbon atom and not the nitrogen atom. The inhibition of bile-salt-dependent lipase does not occur consecutive to the formation of a reactive isocyanate derivative of carbamate but via a tetrahedral intermediate involving essential residues implicated in the enzyme catalytic site. This intermediate evolves by liberation of alcohol (or phenol) and formation of an inactive carbamyl enzyme. Among the carbamates tested, N-butyl-N-methyl-(4-nitrophenyl) carbamate specifically inhibits the bile-salt-dependent lipase; the release of 4-nitrophenol from this carbamate is directly proportional to the enzyme inhibition and it may be defined as a specific active-site titrator for bile-salt-dependent lipases.     

Mechanism of association of a specific aldehyde inhibitor, leupeptin, with bovine trypsin

Leupeptin (acyl peptidyl-L-argininal) is a potent inhibitor of trypsin and related proteases. We analyzed the association of leupeptim with bovine trypsin kinetically, assuming that it proceeds by a pathway which involves two steps: E + I in equilibrium K1 Complex I k-2 in equilibrium k+2 Complex II. The observed dissociation constant (K1) for the first step was 1.24 X 10(-3) M (at pH 8.2 15 degrees C) and the two first-order rate constants (k+2 and k-2) were 166 s-1 and 1.75 X 10(-3.s-1, respectively (at pH 8.2, 15 degrees C). The dissociation constant (Kd) for the whole process was calculated from these parameters to be 1.34 X 10(-8) M. This value is compatible with that determined directly by an independent static method (2.36 X 10(-8) M). We also measured Kd for the leupeptine complex of anhydrotrypsin, a trypsin derivative in which the active-site hydroxyl group is missing. The observed value was about 5 orders of magnitude larger than Kd and was rather similar to K1 in native trypsin. A elupeptin isomer which contains a D-argininal residue did not show strong affinity towards trypsin. These findings suggest that complex II consists of a covalent hemiacetal adduct formed between the serine hydroxyl group in the enzyme active site and the aldehyde group in the inhibitor. The pH dependencies of the dissociation constant and other parameters show that deprotonation of the charge-relay sustem in the active site is important for the formation and stabilization of complex II.     

Slow tight binding inhibition of proteinase K by a proteinaceous inhibitor: conformational alterations responsible for conferring irreversibility to the enzyme-inhibitor complex

The kinetics of slow onset inhibition of Proteinase K by a proteinaceous alkaline protease inhibitor (API) from a Streptomyces sp. is presented. The kinetic analysis revealed competitive inhibition of Proteinase K by API with an IC50 value 5.5 +/- 0.5 x 10-5 m. The progress curves were time-dependent, consistent with a two-step slow tight binding inhibition. The first step involved a rapid equilibrium for formation of reversible enzyme-inhibitor complex (EI) with a Ki value 5.2 +/- 0.6 x 10-6 m. The EI complex isomerized to a stable complex (EI*) in the second step because of inhibitor-induced conformational changes, with a rate constant k5 (9.2 +/- 1 x 10-3 s-1). The rate of dissociation of EI* (k6) was slower (4.5 +/- 0.5 x 10-5 s-1) indicating the tight binding nature of the inhibitor. The overall inhibition constant Ki* for two-step inhibition of Proteinase K by API was 2.5 +/- 0.3 x 10-7 m. Time-dependent dissociation of EI* revealed that the complex failed to dissociate after a time point and formed a conformationally altered, irreversible complex EI**. These conformational states of enzyme-inhibitor complexes were characterized by fluorescence spectroscopy. Tryptophanyl fluorescence of Proteinase K was quenched as a function of API concentration without any shift in the emission maximum indicating a subtle conformational change in the enzyme, which is correlated to the isomerization of EI to EI*. Time-dependent shift in the emission maxima of EI* revealed the induction of gross conformational changes, which can be correlated to the irreversible conformationally locked EI** complex. API binds to the active site of the enzyme as demonstrated by the abolished fluorescence of 5-iodoacetamidofluorescein-labeled Proteinase K. The chemoaffinity labeling experiments lead us to hypothesize that the inactivation of Proteinase K is because of the interference in the electronic microenvironment and disruption of the hydrogen-bonding network between the catalytic triad and other residues involved in catalysis.     

Inhibition of factor Xa by a peptidyl-alpha-ketothiazole involves two steps. Evidence for a stabilizing conformational change.

Recently, peptidylketothiazoles have been shown to be potent inhibitors of proteases, but the details of the interaction have not yet been studied. In the work presented here, the interaction of factor Xa, a coagulation protease, with the transition state inhibitor BnSO(2)-D-Arg-Gly-Arg-ketothiazole (C921-78) is characterized. C921-78 is a tight and selective inhibitor of the coagulation protease factor Xa (K(d) = 14 pM). The hydrolytic activity of factor Xa was inhibited by C921-78 in a time-dependent manner. The rate-limiting step of the bimolecular combination of inhibitor and enzyme was competitive with the substrate. Conversely, the inhibitor could be displaced from the active site of the enzyme after exposure of the preformed complex to an excess of substrate or to the active site inhibitor dansyl-Glu-Gly-Arg-chloromethyl ketone (DEGR-CMK) in a slow reaction. The formation of the C921-78-factor Xa complex resulted in a 60% increase in the magnitude of the fluorescence emission spectrum. Rapid mixing of the enzyme and inhibitor produces a monophasic fluorescence increase, compatible with spectral transition in a single step. The rate constant for this reaction increased hyperbolically with the concentration of C921-78, but the amplitude remained constant. These results are consistent with the initial formation of an enzyme-inhibitor complex (EI), followed by a unimolecular conversion of EI to EI linked to a spectral transition. The rate constants of the isomerization provide an estimate of 300000-fold stabilization. Thus, the inhibition of factor Xa by C921-78 follows a mechanism similar to that described classically for slow tight binding inhibitors. However, the two steps of the reaction cannot be kinetically separated by the rapid equilibrium assumption, and therefore, the formation of EI is partially rate-limiting, too. The driving energy for the unusually fast isomerization step may result from the highly favorable interactions of the inhibitor in the primary binding site.     

Structure-reactivity probes for active site shapes of cholesterol esterase by carbamate inhibitors.

4,4'-Biphenyl-di-N-butylcarbamate (1), (S)-1,1'-bi-2-naphthyl-2, 2'-di-N-butylcarbamate (S-2), (S)-1, 1'-bi-2-naphthyl-2-N-butylcarbamate-2'-butyrate (S-3), 2, 2'-biphenyl-di-N-butylcarbamate (4), 2, 2'-biphenyl-2-N-octadecylcarbamate-2'-N-octylcarbamate (5), 2, 2'-biphenyl-2-N-octadecylcarbamate-2'-N-phenylcarbamate (6), 2, 2'-biphenyl-2-N-butylcarbamate-2'-butyrate (7), 2, 2'-biphenyl-2-N-butylcarbamate-2'-ol (8), 2, 2'-biphenyl-2-N-octylcarbamate-2'-ol (9), (R)-1, 1'-bi-2-N-naphthyl-2-butylcarbamate-2'-ol (R-10), and glyceryl-1,2, 3-tri-N-butylcarbamate (11) are prepared and evaluated for their inhibition effects on porcine pancreatic cholesterol esterase. All inhibitors are irreversible inhibitors of the enzyme. Carbamates 1-3 and 7-10 are the first alkyl chain and esteratic binding site-directed irreversible inhibitors due to the fact that the reactivity of the enzyme is protected by the irreversible inhibitor, trifluoroacetophenone in the presence of these carbamates. Carbamate 1 is the least potent inhibitor for the enzyme probably due to the fact that the inhibitor molecule adopts a linear conformation and one of the carbamyl groups of the inhibitor molecule covalently interacts with the first alkyl chain binding site of the enzyme while the other carbamyl group of the inhibitor molecule exposes outside the active site. With near orthogonal conformations at the pivot bond of biaryl groups, one carbamyl group of carbamates S-2, S-3, and R-10 covalently binds to the first alkyl chain binding site of the enzyme while the other carbamyl, butyryl, or hydroxy group can not bind covalently to the second alkyl chain binding site probably due to the orthogonal conformations. Carbamates 4-9 and 11 are very potent inhibitors for the enzyme probably due to the fact that all these molecules freely rotate at the pivot bond of the biphenyl or glyceryl group and therefore can fit well into the bent-shaped first and second alkyl chains binding sites of the enzyme. Although, carbamates 4-6 and 11 are irreversible inhibitors of cholesterol esterase, the enzyme is not protected but further inhibited by trifluoroacetophenone in the presence of these carbamates. Therefore, carbamates 4-6 and 11 covalently bind to the first alkyl chain binding site of the enzyme by one of the carbamyl groups and may also bind to the second alkyl chain binding site of the enzyme by the second carbamyl group. Besides the bent-shaped conformation, the inhibition by carbamate 6 is probably assisted by a favorable pi-pi interaction between Phe 324 at the second alkyl chain binding site of the enzyme and the phenyl group of the inhibitor molecule. For cholesterol esterase, carbamates 8-10 are more potent than carbamates S-2, 4, and 5 probably due to the fact that the inhibitor molecules interact with the second alkyl chain binding site of the enzyme through a hydrogen bond between the phenol hydroxy group of the inhibitor molecules and the His 435 residue in that site.     

Mechanistic investigation of medium-chain fatty acyl-CoA dehydrogenase utilizing 3-indolepropionyl/acryloyl-CoA as chromophoric substrate analogues.

The CoA derivative 3-indolepropionyl-CoA (IPCoA) serves as a competent pseudosubstrate for the medium-chain fatty acyl-CoA dehydrogenase (MCAD)-catalyzed reaction. The reaction product trans-3-indoleacryloyl-CoA (IACoA) exhibits a characteristic UV-vis absorption spectrum with lambda max = 367 nm and epsilon 367 = 26,500 M-1 cm-1. The chromophoric nature of IACoA allows us to measure the direct conversion of substrate to product (at 367 nm) without recourse to absorption signals for either the enzyme-bound flavin or the coupling electron acceptors, as well as probe the enzyme site environment. The interaction of IACoA with medium chain fatty acyl-CoA dehydrogenase (MCAD)-FAD is characterized by resultant (spectra of the mixture minus the individual components) absorption peaks at 490, 417, and 355 nm. These absorption peaks increase in magnitude as the pH of the buffer media decreases. Transient kinetic analysis for the interaction of MCAD-FAD with IACoA suggests that the formation of the enzyme-IACoA complex proceeds in two steps. The first (fast) step involves the formation of an E-IACoA collision complex, which [formula: see text] is isomerized (concomitant with changes in the protein structure) to an E*-IACoA complex in the second (slow) step. We have studied the effect of pH on Kc, k2, and k-2. While Kc shows practically no dependence on pH (within a 2-fold variation between pH 6.0 and 9.5), k2 and k-2 show a strong dependence on pH. Both k2 and k-2 exhibit a sigmoidal dependence on the pH of the buffer media, with pKa's of 7.53 and 8.30, respectively. In accordance with the model presented herein, the pKa of 7.53 represents an enzyme site group which is involved in the interaction with IACoA within the E-IACoA collision complex. This pKa is perturbed to 8.30 upon isomerization of the collision complex. The pH-dependent changes in k2 and k-2 are such that the equilibrium distribution between E-IACoA and E*-IACoA is favored to the latter complex (by about 20-fold) at lower pH than at higher pH. A cumulative account of the spectral, kinetic, and thermodynamic properties of the enzyme-IACoA complexes has allowed us delineate the microscopic pathway by which the E-IACoA isomerization (presumably via protein conformational changes) is coupled to the proton equilibration steps.     

Inhibition of aminopeptidases by aminophosphonates

More than 30 aminophosphonates were synthesized to probe how the structural changes introduced into the phosphonic acid analogue of leucine, a potent inhibitor of cytosolic leucine aminopeptidase (Giannousis & Bartlett, 1987), affect their ability to inhibit cytosolic (EC 3.4.11.1) and microsomal (EC 3.4.11.2) aminopeptidases. Although most of the compounds studied were found to exert only a modest competitive inhibitory effect, nearly every modification of the structure of the phosphonic acid analogue of leucine was reflected in a marked difference in the affinities of these compounds for the two enzymes. [1-Amino-2-(N-alkylamino)ethyl]phosphonic acids are effective inhibitors of the microsomal enzyme, acting in a time-dependent manner. Kinetic data obtained for these inhibitors correspond to the mechanism for a biphasic slow-binding inhibition process: E + I in equilibrium E* in equilibrium E*I, in which the slow initial isomerization of the enzyme is followed by the fast formation of enzyme-inhibitor complex. The most effective inhibitor of this type was [1-amino-2-(N-cyclohexylamino)ethyl]phosphonic acid, which has a Ki value of 0.87 microM toward the microsomal aminopeptidase--a value that can be considered as equipotent with bestatin and with leucinal and hydroxamic acids, the strongest known nonpeptide inhibitors of this enzyme.     

Slow-tight binding inhibition of xylanase by an aspartic protease inhibitor: kinetic parameters and conformational changes that determine the affinity and selectivity of the bifunctional nature of the inhibitor

The first report of slow-tight inhibition of xylanase by a bifunctional inhibitor alkalo-thermophilic Bacillus inhibitor (ATBI), from an extremophilic Bacillus sp. is described. ATBI inhibits aspartic protease (Dash, C., and Rao, M. (2001) J. Biol. Chem., 276, 2487-2493) and xylanase (Xyl I) from a Thermomonospora sp. The steady-state kinetics revealed time-dependent competitive inhibition of Xyl I by ATBI, consistent with two-step inhibition mechanism. The inhibition followed a rapid equilibrium step to form a reversible enzyme-inhibitor complex (EI), which isomerizes to the second enzyme-inhibitor complex (EI*), which dissociated at a very slow rate. The rate constants determined for the isomerization of EI to EI*, and the dissociation of EI* were 13 +/- 1 x 10(-6) s(-1) and 5 +/- 0.5 x 10(-8) s(-1), respectively. The K(i) value for the formation of EI complex was 2.5 +/- 0.5 microm, whereas the overall inhibition constant K(i)* was 7 +/- 1 nm. The conformational changes induced in Xyl I by ATBI were monitored by fluorescence spectroscopy and the rate constants derived were in agreement with the kinetic data. Thus, the conformational alterations were correlated to the isomerization of EI to EI*. ATBI binds to the active site of the enzyme and disturbs the native interaction between the histidine and lysine, as demonstrated by the abolished isoindole fluorescence of o-phthalaldehyde (OPTA)-labeled Xyl I. Our results revealed that the inactivation of Xyl I is due to the disruption of the hydrogen-bonding network between the essential histidine and other residues involved in catalysis and a model depicting the probable interaction between ATBI or OPTA with Xyl I has been proposed.     

Fructose 1,6-bisphosphate aldolase from rabbit muscle. The isomerization of the enzyme-dihydroxyacetone phosphate complex.

The formation and dissociation of the aldolase-dihydroxyacetone phosphate complex were studied by following changes in A240 [Topper, Mehler & Bloom (1957), Science 126, 1287-1289]. It was shown that the enzyme-substrate complex (ES) slowly isomerizes according to the following reaction: (formula: see text) the two first-order rate constants for the isomerization step being k+2 = 1.3s-1 and k-2 = 0.7s-1 at 20 degrees C and pH 7.5. The dissociation of the ES complex was provoked by the addition of the competitive inhibitor hexitol 1,6-bisphosphate. At 20 degrees C and pH 7.5, k+1 was 4.7 X 10(6)M-1-S-1 and k-1 was 30s-1. Both the ES and the ES* complexes react rapidly with 1.7 mM-glyceraldehyde 3-phosphate, the reaction being practically complete in 40 ms. This shows that the ES* complex is not a dead-end complex. Evidence was also provided that aldolase binds and utilizes only the keto form of dihydroxyacetone phosphate.     

Serine protease mechanism: structure of an inhibitory complex of alpha-lytic protease and a tightly bound peptide boronic acid

The structure of the complex formed between alpha-lytic protease, a serine protease secreted by Lysobacter enzymogenes, and N-tert-butyloxycarbonylalanylprolylvaline boronic acid (Ki = 0.35 nM) has been studied by X-ray crystallography to a resolution of 2.0 A. The active-site serine forms a covalent, nearly tetrahedral adduct with the boronic acid moiety of the inhibitor. The complex is stabilized by seven hydrogen bonds between the enzyme and inhibitor with additional stabilization arising from van der Waals interactions between enzyme and inhibitor side chains and the burying of 330 A2 of hydrophobic surface area. Hydrogen bonding between Asp-102 and His-57 remains intact in the enzyme-inhibitor complex, and His N epsilon 2 is well positioned to donate its hydrogen to the leaving group. Little change in the positions of protease residues was observed on complex formation (root mean square main chain deviation = 0.13 A), suggesting that in its native state the enzyme is complementary to tetrahedral reaction intermediates or to the nearly tetrahedral transition state for the reaction.     

pH-dependence and structure–activity relationships in the papain-catalysed hydrolysis of anilides

The pH-dependence of the Michaelis-Menten parameters for the papain-catalysed hydrolysis of N-acetyl-l-phenylalanylglycine p-nitroanilide was determined. The equilibrium binding constant, K(s), is independent of pH between 3.7 and 9.3, whereas the acylation constant, k(+2), shows bell-shaped pH-dependence with apparent pK(a) values of 4.2 and 8.2. The effect of substituents in the leaving group on the acylation constant of the papain-catalysed hydrolysis of hippuryl anilides and N-acetyl-l-phenylalanylglycine anilides gives rise in both series to a Hammett rho value of -1.04. This indicates that the enzyme provides electrophilic, probably general-acid, catalysis, as well as the nucleophilic or general-base catalysis previously found. A mechanism involving a tetrahedral intermediate whose formation is general-base-catalysed and whose breakdown is general-acid-catalysed seems most likely. The similarity of the Hammett rho values appears to exclude facilitated proton transfer as a means through which the specificity of papain is expressed.