Predicting enzyme behavior in nonconventional media: correlating nitrilase function with solvent properties

The insolubility of nitrile substrates in aqueous reaction mixture decreases the enzymatic reaction rate. We studied the interaction of fourteen water miscible organic solvents with immobilized nitrile hydrolyzing biocatalyst. Correlation of nitrilase function with physico-chemical properties of the solvents has allowed us to predict the enzyme behavior in such non-conventional media. Addition of organic solvent up to a critical concentration leads to an enhancement in reaction rate, however, any further increase beyond the critical concentration in the latter leads to the decrease in catalytic efficiency of the enzyme, probably due to protein denaturation. The solvent dielectric constant (epsilon) showed a linear correlation with the critical concentration of the solvent used and the extent of nitrile hydrolysis. Unlike alcohols, the reaction rate in case of aprotic solvents could be linearly correlated to solvent log P. Further, kinetic analysis confirmed that the affinity of the enzyme for its substrate (K (m)) was highly dependent upon the aprotic solvent used. Finally, the prospect of solvent engineering also permitted the control of enzyme enantioselectivity by regulating enantiomer traffic at the active site.     

Modification and inactivation of rhodanese by 2,4,6-trinitrobenzenesulphonic acid

Bovine liver rhodanese (thiosulphate sulphurtransferase, EC 2.8.1.1) is modified by 2,4,6-trinitrobenzenesulphonic acid, by the use of modifying agent concentrations in large excess over enzyme protein concentration. The end-point of the reaction, viz., the number, n, per enzyme protein molecule, of modifiable amino groups was determined graphically by the Kézdy-Swinbourne procedure. It was found that the value for n depends on the pH of the reaction medium, and ranges from 2, at pH 7.00, to 10.66, at pH 9.00. Again, the value for n increases with an increase in the concentration of 2,4,6-trinitrobenzenesulphonic acid used, with values ranging from 3.52, at 0.10 mM modifying agent, to 8.96, at 2 mM modifying agent. Rhodanese primary amino groups modification by 2,4,6-trinitrobenzenesulphonic acid is described by a summation of exponential functions of reaction time at pH values of 8.00 or higher, while at lower pH values it is described by a single exponential function of reaction time. However, the log of the first derivative, at initial reaction conditions, of the equation describing protein modification, is found to be linearly dependent on the pH of the reaction. An identical linear dependence is also found when the log of the first derivative, at the start of the reaction, of the equation describing modification-induced enzyme inactivation is plotted against the pH values of the medium used. In consequence, the fractional concentration of rhodanese modifiable amino groups essential for enzyme catalytic function is equal to unity at all reaction pH values tested. It is accordingly concluded that, when concentrations of 2,4,6-trinitrobenzenesulphonic acid in excess of protein concentration are used, all rhodanese modifiable amino groups are essential for enzyme activity. A number of approaches were used in order to establish a mechanism for the modification-induced enzyme inactivation observed. These approaches, all of which proved to be negative, include the possible modification of enzyme sulfhydryl groups, disulphide bond formation, enzyme inactivation due to sulphite released during modification, modification-induced enzyme protein polymerization, syncatalytic enzyme modification and hydrogen peroxide-mediated enzyme inactivation.     

EFFECT OF GTP AND OTHER NUCLEOSIDE TRIPHOSPHATES ON GMP INCORPORATION BY ISOLATED CORTICAL NUCLEI

—The concentration of GTP was found to be critically important in determining the characteristics of incorporation of GMP by DNA-dependent RNA polymerase from rat brain nuclei. The linearity of the incorporation rate was related to the log of the GTP concentration. Three hundred μM-GTP in the presence of the other nucleoside triphosphates (1 mM) was near to the optimal conditions in terms of maximum incorporation and linearity. The concentration of ammonium sulfate was an important factor in determining the maximum GMP and UMP incorporation. The U/G incorporation ratio was less than one at low concentrations of substrate and increased with increasing substrate or ammonium sulfate concentration. α-Amanitin strongly inhibited the reaction, indicating that RNA polymerase II is the effective enzyme.     

The influence of glucose and ethanol on enzymic hydrolysis of steam-exploded bagasse

summary The rate of enzymic hydrolysis of steam-exploded bagasse was found to decrease linearly with increasing concentration of glucose and ethanol, with complete cessation of reaction predicted in the presence effects of glucose and ethanol were found to be additive. The significantly greater tolerance of the enzyme to ethanol can be utilised in the simultaneous hydrolysis and fermentation of bagasse cellulose to improve hydrolysis rate.     

One explanation for the variability of the bacterial suspension test

Disinfection kinetic studies of sodium dodecyl sulphate, benzalkonium chloride and sodium hypochlorite against Staphylococcus aureus revealed that when a higher inoculum level of Staph. aureus than normal was used (approximately 1 log higher), the efficacy of disinfection was severely attenuated. Kinetic analysis using the Hom model for experiments carried out on tests using 3 x 108 organisms ml-1 were unable to account for the large increase in disinfection power observed when smaller inoculum levels were used. Since the inoculum was the same in every way except for the numbers used, the large variations in the log reduction/time curves could not be explained by a variation in the resistance of the population to the biocide, as identical log reduction-time curves should have resulted. The level of disinfection achieved for a given concentration of biocide was found to be approximately linearly related to the cell number ml-1 of test solution and not to the log number. The variation observed is believed to occur due to intrinsic self-quenching of the biocide by the microbes during the course of the disinfection test. As the level of free biocide decreases, the rate of reaction decreases, giving the tails of the log reduction/time curves. Such intrinsic self-quenching could explain the large variations known to occur in the legally required disinfection suspension tests.     

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.     

消炎痛对猪胃H~＋／K~＋－ATP_（ase）抑制的动力学

消炎痛作为一种可引起胃粘膜急性病变的药物,用分离提纯的猪胃Ｈ＋／Ｋ＋－ＡＴＰａｓｅ证明,它可以显著的抑制此酶的活力,０．１ｍｇ／ｍＬ时即可抑制酶活力２７％,０．５ｍｇ／ｍＬ时可抑制全部活力,其Ｋ（０．５）为０．１８ｍｇ／ｍＬ。消炎痛对Ｈ＋／Ｋ＋－ＡＴＰａｓｅ的抑制随３０℃时预保温时间的延长而加剧,１０ｍｉｎ预保温可抑制总活力的５０％。消炎痛并不影响Ｈ＋／Ｋ＋－ＴＡＰａｓｅ的转换温度（３９℃）以及最适ｐＨ（约ｐＨ７．５）,但酸性条件下消炎痛对Ｈ＋／Ｋ＋－ＡＴＰａｓｅ抑制比碱性条件下强烈。在我们的实验条件下,消炎痛对Ｈ＋／Ｋ＋－ＡＴＰａｓｅ的抑制与Ｈ＋／Ｋ＋－ＡＴＰａｓｅ量成正比,它不影响酶的Ｋｍ值（０．１１ｍｍｏｌ／Ｌ）,而是显著降低Ｖｍａｘ,因而它是此酶的可逆性非竞争性抑制剂,其Ｋｉ为０．３２ｍｍｏｌ／Ｌ。     

The binding of a K+ competitive ligand, 2-methyl,8-(phenylmethoxy)imidazo(1,2-a)pyridine 3-acetonitrile, to the gastric (H+ + K+)-ATPase

2-Methyl,8-(phenylmethoxy)imidazo(1,2-a)pyridine 3-acetonitrile (SCH 28080) is a freely reversible K+ site inhibitor of the gastric (H+ + K+)-ATPase. In the presence of 2 mMMgSO4, [14C]SCH 28080 bound saturably to gastric vesicle preparations containing the (H+ + K+)-ATPase and was displaced by lumenal K+. A binding stoichiometry of 2.2 +/- 0.1 mol of SCH 28080/mol of catalytic phosphorylation sites was observed. The affinity of SCH 28080 binding was increased approximately 10-fold (to 45 nM) in the presence of 2 mM ATP. High affinity binding also occurred with 2 microM ATP but not with up to 200 microM D-[beta, gamma-CH2]ATP, suggesting that high affinity binding was to a phosphorylated form of the enzyme. In the presence of ATP, the association rate constant was linearly related to the concentration of SCH 28080. However, the association and dissociation rates of SCH 28080 binding were slow, especially at low temperature (at 1.5 degrees C half-maximal binding of 50 nM SCH 28080 was calculated to occur after 232 s). Binding appeared to be predominantly entropy driven with a high activation energy (40 kJ/mol at 37 degrees C). In the absence of ATP, the association rate constant was not linearly related to the concentration of SCH 28080, suggesting that a conformational change in the enzyme was required before binding could occur.     

Analysis and quantification of a mixed exo-acting and endo-acting polysaccharide depolymerization system

A new mathematical model has been proposed based on a model presented by Suga, van Dedem, and Moo-Young.(10) The model requires a separate differential equation for each polymeric species (differentiated by degree of polymerization) in the reaction mixture. The main contribution of this model is the incorporation of experimental molecular weight distributions as the initial conditions. These molecular weight distributional as the initial conditions were obtained using modern analytical equipment previouly unknown for this application. The equipment, SEC/LALLS, measures relative concentrations of specific molecular weight species along with the corresponding molecular weights, thus yielding (through some mathematical manipulation) the absolute concentration of each molecular weight species. The concentration at each molecular weight can then be incorporated as the initial condition for that equation. Theoretically, the system of differential equations can be solved to give a more realistic time course of reaction.Synergism between endo-acting and exo-acting enzymes was examined theoretically using the mathematical model. Through model predictions, it was found that synergy is based on two fundamental parameters: (1) each enzyme's activity relative to the sum of enzyme activities and, (2) overall substrate concentration relative to the exo-acting enzyme's Michaeiis kinetic constant K(m). Theoretically, synergism increases as a function of reaction time. Intermediate endo fractions (ratio of endo-acting enzyme activity to the sum of endo-acting and exo-acting enzyme activity) from 0.3 to 0.7 exhibit the most synergism. Values of k[log(K(m, exo)/S(0))] above about zero also exhibits the most synergism.An examination of experimental data obtained both by SEC/LALLS and by reducing sugar measurements shows that the model is inadequate for successfully predicting quantities associated with the substrate during reaction. This is especially true for synergism predictions. At short reaction times, the model predicts the data fairly well, but at longer times the predictions are inconsistent with experimental data. These inconsistencies may be due to complicating phenomena such as enzyme inhibitions.     

The effect of K+ concentration on the energy metabolism in perfused rat heart

From experiments at various perfusion pressures in hemoglobin-free perfused rat hearts, oxygen consumption and redox shift of pyridine nucleotide were found to vary linearly with cardiac work. This relation was used for analysis of the energy metabolism associated with ion pumps. Mechanical activities such as left ventricular pressure and heart rate varied with the extracellular K+ concentration. Ion-pump dependent changes in oxygen consumption and redox state of pyridine nucleotide, estimated as the difference of the values at normal (4.7 mM) and various other extracellular K+ concentrations with corrections for the change due to mechanical work, were found to vary linearly with the K+ concentration. The slope for oxygen consumption was about 0.1 mumol/min/g X wet wt per mM K+. Lactate release changed markedly but transiently, about 1 min after changing the extracellular K+ concentration, and its amount varied linearly with the K+ concentration. In the steady state, however, lactate release was almost independent of the extracellular K+ concentration, although oxidized pyridine nucleotide increased with increasing K+ concentration. Coronary flow increased with the extracellular K+ concentration. Heart rate changed little between 1 and 12 mM K+, but decreased sharply above 12 mM K+. At 20 mM K+, heart beat was arrested and approximately 40% of myoglobin was deoxygenated. The intracellular oxygen concentration was estimated to be about 10 microM even during aerobic perfusion. Similarly, Ca2+-free arrested heart was found to be in a hypoxic state. The results showed that oxygen entry into cardiac tissue is facilitated by the cardiac cycle.     

Creatine kinase equilibrium and lactate content compared with muscle pH in tissue samples obtained after isometric exercise

Muscle biopsies taken from the musculus quadriceps femoris of man were analysed for pH, ATP, ADP, AMP, creatine phosphate, creatine, lactate and pyruvate. Biopsies were taken at rest, after circulatory occlusion and after isometric contraction. Muscle pH decreased from 7.09 at rest to 6.56 after isometric exercise to fatigue. Decrease in muscle pH was linearly related to accumulation of lactate plus pyruvate. An increase of 22mumol of lactate plus pyruvate per g of muscle resulted in a fall of 0.5pH unit. The apparent equilibrium constant of the creatine kinase reaction (apparent K(CK)) increased after isometric contraction and a linear relationship between log(apparent K(CK)) and muscle pH was obtained. The low content of creatine phosphate in muscle after contraction as analysed from needle-biopsy samples is believed to be a consequence of an altered equilibrium state of the creatine kinase reaction. This in turn is attributed mainly to a change in intracellular pH.     

The comparison of the estimation of enzyme kinetic parameters by fitting reaction curve to the integrated Michaelis-Menten rate equations of different predictor variables

The estimation of enzyme kinetic parameters by nonlinear fitting reaction curve to the integrated Michaelis-Menten rate equation ln(S(0)/S)+(S(0)-S)/K(m)=(V(m)/K(m))xt was investigated and compared to that by fitting to (S(0)-S)/t=V(m)-K(m)x[ln(S(0)/S)/t] (Atkins GL, Nimmo IA. The reliability of Michaelis-Menten constants and maximum velocities estimated by using the integrated Michaelis-Menten equation. Biochem J 1973;135:779-84) with uricase as the model. Uricase reaction curve was simulated with random absorbance error of 0.001 at 0.075 mmol/l uric acid. Experimental reaction curve was monitored by absorbance at 293 nm. For both CV and deviation <20% by simulation, K(m) from 5 to 100 micromol/l was estimated with Eq. (1) while K(m) from 5 to 50 micromol/l was estimated with Eq. (2). The background absorbance and the error in the lag time of steady-state reaction resulted in negative K(m) with Eq. (2), but did not affect K(m) estimated with Eq. (1). Both equations gave better estimation of V(m). The computation time and the goodness of fit with Eq. (1) were 40-fold greater than those with Eq. (2). By experimentation, Eq. (1) yielded K(m) consistent with the Lineweaver-Burk plot analysis, but Eq. (2) gave many negative parameters. Apparent K(m) by Eq. (1) linearly increased, while V(m) were constant, vs. xanthine concentrations, and the inhibition constant was consistent with the Lineweaver-Burk plot analysis. These results suggested that the integrated rate equation that uses the predictor variable of reaction time was reliable for the estimation of enzyme kinetic parameters and applicable for the characterization of enzyme inhibitors.     

Papain hydrolysis of X-phenyl-N-methanesulfonyl glycinates: a quantitative structure-activity relationship and molecular graphics analysis

The hydrolysis of 32 X-phenyl-N-methanesulfonyl glycinates by papain was investigated. It was found that the variation in the Michaelis constants could be rationalized by the following correlation equation: log 1/Km = 0.61 pi '3 + 0.46 MR4 + 0.55 sigma + 2.00 with a correlation coefficient of 0.945. In this expression, pi '3 is the hydrophobic constant for the more lipophilic of the two possible meta substituents, MR4 is the molar refractivity of 4-substituents, and sigma is the Hammett constant summed for all substituents. Using this equation, we designed, synthesized, and successfully predicted Km for a new congener intended to maximize binding (1/Km). The interactions involved in enzyme-substrate binding, as characterized by the correlation equation, are interpreted using a computer-constructed color three-dimensional-graphics molecular model of the enzyme active site. The nonenzymatic hydrolysis (both acid and basic) of phenyl hippurates yield rate constants which are well correlated by Hammett equations; however, log k for both acid and alkaline hydrolysis are not linearly related to log 1/Km or log kcat/Km.     

Kinetics of chitinase production. I. Chitin hydrolysis

As part of the development of a comprehensive mathematical model for chitinase production by Serratia marcescens QMB 1466 growing on chitin, the different mass transport and kinetic steps involved during chitin hydrolysis were studied. The experimental results for the hydrolysis of chitin by a crude preparation of chitinase show a system kinetically limited by the overall rate of chitin hydrolysis. This rate is linearly related to the concentration of enzyme adsorbed on the chitin particle. Adsorbed and bulk enzyme concentration were found to be related through a Langmuir type of isotherm.     

Detection of ouabain-insensitive H(+)-transporting, K(+)-stimulated p-nitrophenylphosphatase activity in rat gastric glands by cerium-based cytochemistry

We employed a modification of our previously reported cerium-based cytochemical method for ouabain-sensitive, K-dependent p-nitrophenylphosphatase (Na-K ATPase) activity to detect ouabain-insensitive, K-stimulated p-nitrophenylphosphatase (K-pNPPase) activity in rat gastric glands. Biochemically, the enzyme activity of gastric glands incubated in a medium containing 50 mM Tricine buffer (pH 7.5), 50 mM KCl, 10 mM MgCl2, 2 mM CeCl3, 2 mM p-nitrophenylphosphate (pNPP), 2.5 mM levamisole, 10 mM ouabain, and 0.00015% Triton X-100, was optimal at pH 7.5-8.0 and decreased above pH 8.5. The amount of p-nitrophenol after incubation increased linearly in proportion to the amount of tissue in the medium. The enzyme activity was inhibited by omeprazole, sodium flouride (NaF), N-ethylmaleimide (NEM), and dicyclohexylcarbodiimide (DCCD). Heat-treated specimens had no enzyme activity. The enzyme activity increased with addition of K ions up to the concentration of 50 mM, and became constant above 50 mM. Cytochemically, the parietal cells of the gastric glands reacted positively for ouabain-insensitive K-pNPPase activity. Intense reaction was observed at the microvilli of the luminal surface and the intracellular canaliculi. The tubulovesicular system showed weak enzyme activity. The reaction products were found as fine, granular, electron-dense deposits in the cytoplasm just beneath the plasma membrane. The ouabain-insensitive K-pNPPase activity detected in this study appears, therefore, to be associated with that of H-transporting, K-stimulated adenosine triphosphatase (H-K ATPase).     

Hydrolysis of rice bran oil using immobilized lipase in a stirred batch reactor

Candida cylindracea lipase was immobilized by adsorption on acid washed glass beads. It was observed that protein loading of the support depends on the size of the particle, with smaller particle containing higher amount of protein per unit weight. Initial reaction rate linearly varied up to enzyme concentration of 17.25 U/mL. Amount of free fatty acids produced was linearly proportional up to the enzyme loading of 1650 μg/g of bead. Achievement of chemical equilibrium took longer time in the case of less protein loading. Degree of hydrolysis was found to decrease in second and third consecutive batch operations on repeated use of immobilized lipase.     

Transcuticular solute movement in parasitic nematodes: relationship between non-polar solute transport and partition coefficient

The rate constants (K) for steroid transport across the isolated cuticle of Ascaris suum were, linearly related to partition coefficients (P) of steroids in model solvents. The slope of log P vs log K for an isolated cuticle was closest to unity for the more polar solvents, octanol and ether, suggesting that the barrier to steroid movement across the cuticle has partition properties similar to these solvents. The transcuticular movement of steroids into intact adult A. suum and infective larvae of Haemonchus contortus was predicted by the P in model solvents suggesting that the same barriers for transcuticular solute movement may exist in intact worms as in isolated cuticles.     

Studies on the kinetic effects of adenosine-3':5'-monophosphate-dependent phosphorylation of purified pig-liver pyruvate kinase type L.

The effect of cyclic-AMP-dependent phosphorylation on the activity of isolated pig liver pyruvate kinase was studied. It was found that the major kinetic effect of the phosphorylation was to reduce the affinity for the substrate phosphoenolpyruvate, K0.5 for this substrate increasing from 0.3 to 0.9 mM upon phosphorylation. The cooperative effect with phosphoenolpyruvate was enhanced, the Hill constant nH increasing concomitantly from 1.1 to 1.5. V was unaltered. The change in activity occurred in parallel with the phosphate incorporation, except during the initial part of the reaction, when inactivation was correspondingly slower. The affinity for the second substrate ADP was unchanged, with an apparent Km of 0.3 mM at saturating concentration of phosphoenolpyruvate. Likewise, the requirement for potassium was unaffected, whereas the phosphoenzyme required a higher concentration of magnesium ions for maximal activity, compared with the control enzyme. The inhibitory effect of the phosphorylation was counteracted by positive effectors, fructose 1,6-biphosphate in micromolar concentrations completely activated the phosphoenzyme, resulting in an enzyme with properties similar to the fructose 1,6-biphosphate-activated unphosphorylated enzyme, with K0.5 for phosphoenolpyruvate about 0.025 mM and with a Hill constant of 1.1. Hydrogen ions were also effective in activating the phosphoenzyme. Thus, when pH was lowered from 8 to 6.5 the inhibition due to phosphorylation was abolished. The phosphoenzyme was sensitive to further inhibition by negative effectors such as ATP and alanine. 2 mM ATP increased K0.5 for phosphoenolpyruvate to 1.5 mM and nH to 2.3. The corresponding values with alanine were 1.3 mM and 1.9. Phosphorylation is thought to be an additional mechanism of inhibition of the enzyme under gluconeogenetic conditions.     

Quantitative structure-activity relationships for the pre-steady state acetylcholinesterase inhibition by carbamates

4-Nitrophenyl-N-substituted carbamates (1) are characterized as pseudosubstrate inhibitors of acetylcholinesterase. The first step is formation of the enzyme-inhibitor tetrahedral intermediate with the inhibition constant (Ki), the second step is formation of the carbamyl enzyme with the carbamylation constant (kc), and the third step is hydrolysis of the carbamyl enzyme with decarbamylation constant (kd). According to pre-steady state kinetics the Ki step is divided further into two steps: (1) formation of the enzyme-inhibitor complex with the dissociation constant (KS) and (2) formation of the enzyme-inhibitor tetrahedral intermediate from the complex with the equilibrium constant (k2/k-2). Since the inhibitors are protonated in pH 7.0 buffer solution, the virtual dissociation constant (KS') of the enzyme-protonated inhibitor complex can be calculated from the equation, -log KS'=-log KS-pKa + 14. The -logKS, -log KS', log k2, and log k-2 values are multiply linearly correlated with the Jave equation (log(k/k0)=rho*sigma* + deltaEs + psi pi). For -log KS'-sigma*-Es)pi-correlation, the rho* value of -0.4 indicates that the enzyme-protonated inhibitor complexes have more positive charges than the protonated inhibitors, the delta value of 0.44 suggests that the bulkily substituted inhibitors lessen the reaction due to the difficulty of the inhibitors to enter the narrow enzyme active site gorge, and the psi value of 0.27 implies that the inhibitors with hydrophobic substituents accelerate the inhibitors entering the active site gorge of the enzyme. For log k2/k-2,-sigma*-Es-pi-correlation, the rho* value of 1.1 indicates that the enzyme-protonated inhibitor tetrahedral intermediates have more negative charges than the enzyme-protonated inhibitor complexes, the delta value of 0.15 suggests that the bulkily substituted inhibitors are difficult to bind into a small acyl binding site of the enzyme, and the psi value of -0.3 implies that the inhibitors with hydrophobic substituents resist binding to the hydrophilic acyl binding site of the enzyme.