Kinetic studies of wheat carboxypeptidase-catalyzed reaction: differences in pressure and temperature dependence of peptidase and esterase activities

A kinetic study of hydrolytic catalysis by wheat bran carboxypeptidase (carboxypeptidase W) was carried out using 3-(2-furyl)acryloyl-acylated (Fua-) synthetic substrates. This enzyme showed high esterase activity in addition to the intrinsic carboxypeptidase activity. The optimum pH for the peptidase activity (kcat/Km) was at pH 3.3 and the kcat/Km value decreased with increasing pH with an apparent pKa of 4.50, while the esterase activity increased with pH up to pH 8 with an apparent pKa of 6.04. Optimum pH's for kcat for the peptidase and esterase reactions were also very different and their apparent pKa values were 3.80 and 6.15, respectively. From a measurement of the pressure dependences of kcat and Km, the activation volumes (delta V not equal to) and reaction volumes (delta V), respectively, were determined. delta V not equal to for kcat was -7 to -8 ml/mol for peptidase and -2 to -3 ml/mol for esterase. These results lead us to propose that the peptidase and esterase activities of carboxypeptidase W are different not in the rate-determining steps in a common reaction pathway, but in the binding modes and/or catalytic site(s).     

Pressure effects on substrate activation phenomena in the alpha-chymotrypsin-catalyzed hydrolysis of p-nitrophenyl acetate

With and without p-chlorophenol as an activator, the rates of hydrolysis of p-nitrophenyl acetate catalyzed by alpha-chymotrypsin were measured at pressures up to 2 kbar at 25 degrees C. From the pressure dependence of the rate constant (kcat)A and (kcat)0 of the product formation with and without an activator, the activation volumes (delta V not equal to cat)A and (delta not equal to cat)0 were +2 and -6 +/- 1 cm3.mol-1. From the pressure dependence of the equilibrium constant (KA) of incorporation of p-chlorophenol into the enzyme, the volume change (delta VA) was -10 +/- 1 cm3.mol-1. The mechanisms of the substrate activation are discussed in terms of the activation and reaction volumes.     

High-pressure effect on the equilibrium and kinetics of cyanide binding to chloroperoxidase

The kinetics of cyanide binding to chloroperoxidase were studied using a high-pressure stopped-flow technique at 25 degrees C and pH 4.7 in a pressure range from 1 to 1000 bar. The activation volume change for the association reaction is delta V not equal to + = -2.5 +/- 0.5 ml/mol. The total reaction volume change, determined from the pressure dependence of the equilibrium constant, is delta V degrees = -17.8 +/- 1.3 ml/mol. The effect of temperature was studied at 1 bar yielding delta H not equal to + = 29 +/- 1 kJ/mol, delta S not equal to + = -58 +/- 4 J/mol per K. Equilibrium studies give delta H degrees = -41 +/- 3 kJ/mol and delta S degrees = -59 +/- 10 J/mol per K. Possible contributions to the binding process are discussed: changes in spin state, bond formation and conformation changes in the protein. An activation volume analog of the Hammond postulate is considered.     

Temperature-dependent effects of high pressure on the bioluminescence of firefly luciferase.

This study measured the effect of high pressure on the enzyme kinetics of firefly luciferase. When firefly luciferase is mixed with luciferin and ATP, a transient flash of light is produced, followed by a weak light, lasting hours. The first stage reaction produces an enzyme-luciferin-AMP complex and pyrophosphate. Addition of pyrophosphate to the reaction mixture decelerated the reaction rate, and the initial flash was prolonged to a plateau, showing a quasi-equilibrium state. The effects of temperature and pressure were analyzed at the plateau. The temperature scan showed that the maximum light intensity was observed at about 22.5 degrees C. When pressurized below the temperature optimum, pressure decreased the light intensity, while increasing it above the temperature optimum. According to the theory of absolute reaction rate, the following values were obtained for the bioluminescent reaction: delta V++ = 823.7 - 2.8 T cm3/mol and delta V = -280.47 + 0.94T cm3/mol, where T is the absolute temperature, delta V++ and delta V are, respectively, activation volume and the volume change due to thermal unfolding. The optimal temperature for the maximum light output occurs because the reaction rate increases with the temperature elevation at low temperature range, but the thermal unfolding of the enzyme decelerates the reaction velocity when the temperature exceeds a critical value. The intensity of luminescence is modified by the influence of pressure on both delta V++ and delta V. So long as the volume of the activated complex (V++) exceeds the average volume of the nonactivated complex (VN), pressure will slow down the reaction. At the point where the volumes become equal, there is no change in the rate under pressure. When the volume of the activated complex is less than that of the reactants, pressure will speed up the rate. This study showed that firefly luciferase is not exceptional to other enzymes in responding to high pressure.     

Effects of high pressure on the single-turnover kinetics of the carbamylation of cholinesterase

Pressure, as a perturbing variable, is one of the most powerful tools to investigate the thermodynamic parameters of chemical reactions and to study the mechanism of enzyme-catalyzed reactions. The effect of elevated hydrostatic pressure (up to 0.8 kbar) on the reaction of butyrylcholinesterase with N-methyl-(7-dimethylcarbamoxy)quinolinium was determined under single-turnover conditions at 35 degrees C. The rate of carbamylation was monitored as the accumulation of the fluorescent ion, N-methyl-7-hydroxyquinolinium, in a high-pressure stopped-flow apparatus designed for the assay of fluorescence. Elevated pressure favored formation of the enzyme-substrate complex but inhibited carbamylation of the enzyme. Because a single reaction step was recorded, it was possible to interpret the data obtained under high pressure in the form of Michaelis-Menten equations. From the pressure dependence of the dissociation constant for the enzyme-substrate complex and the rate constant for carbamylation, maximal volume changes accompanying these events were determined. The value for the binding process, delta Vb = -129 ml.mol-1, is too large to be related only to volumetric changes in the active center. Substrate-induced conformational change and change of water structure appear to be the dominant contributions to the overall volume change associated with substrate binding. The large positive activation volume measured (delta V not equal to = 119 ml.mol-1) may also reflect extended structural and hydration changes. At pressures greater than 0.4 kbar, an additional pressure effect, dependent on substrate concentration, occurred in a narrow pressure interval. This effect may have resulted from a substrate-induced pressure-sensitive enzyme conformational state.     

Effects of pressure and temperature on the reactions of horseradish peroxidase with hydrogen cyanide and hydrogen peroxide.

Reactions of ferric horseradish peroxidase with hydrogen cyanide and hydrogen peroxide were studied as a function of pressure. Activation volumes are small and differ in sign (delta V = 1.7 +/- 0.5 ml/mol for peroxidase + HCN and -1.5 +/- 0.5 ml/mol for peroxidase + H2O2). The temperature dependence of cyanide binding to horseradish peroxidase was also determined. A comparison is made of relevant parameters for cyanide binding and compound I formation.     

High-pressure laser photolysis study of hemoproteins. Effects of pressure on carbon monoxide binding dynamics for R- and T-state hemoglobins

The bimolecular association reaction of carbon monoxide to human adult hemoglobin at pH 7, 20 degrees C, was examined as a function of pressure up to 1500 bar by means of high-pressure laser photolysis. The apparent quantum yield for a millisecond recombination reaction decreased with pressure, which was attributed to an increase in the fraction of nanosecond geminate recombination reaction. On the basis of the pressure dependence of the recombination rate, the activation volumes at normal pressure for the binding of carbon monoxide to the R- and T-state hemoglobins were determined as -9.0 +/- 0.7 and -31.7 +/- 2.4 cm3 mol-1, respectively. Since the activation volumes for the overall CO association reaction were negative, it seems that the iron-ligand bond formation process mainly contributes to the rate-limiting step for both quaternary structures. The characteristic pressure dependence of the activation volume was observed for the R-state Hb but not for the T-state Hb. At 1000 bar, the activation volume for the R-state Hb was reduced to nearly zero, probably resulting from the contribution of the ligand migration process to the rate-limiting step. The effect of pressure on the activation enthalpy and entropy was also extracted from the data.     

Unusual pressure effects on ligand rebinding to the human myoglobin Leucine 29 mutants

Using high pressure flash photolysis, we revealed that the side chain of Leu(29) controls the reaction volume of the ligand migration process in myoglobin, which is the primary factor for the unusual activation volume of ligand binding in some Leu(29) mutants. As we previously reported (Adachi, S., Sunohara, N., Ishimori, K., and Morishima, I. (1992) J. Biol. Chem. 267, 12614-12621), CO bimolecular rebinding in the L29A mutant was unexpectedly decelerated by pressurization, suggesting that the rate-determining step is switched to ligand migration. However, very slow CO bimolecular rebinding of the mutants implies that bond formation is still the rate-determining step. To gain further insights into effects of the side chain on ligand binding, we prepared some new Leu(29) mutants to measure the CO and O(2) rebinding reaction rates under high hydrostatic pressure. CO bimolecular rebinding in the mutants bearing Gly or Ser at position 29 was also decelerated upon pressurization, resulting in apparent positive activation volumes (DeltaV), as observed for O(2) binding. Based on the three-state model, we concluded that the increased space available to ligands in these mutants enhances the volume difference between the geminate and deoxy states (DeltaV(32)), which shifts the apparent activation volume to the positive side, and that the apparent positive activation volume is not due to contribution of the ligand migration process to the rate-determining step.     

Soman inhibition of butyrylcholinesterase in the presence of substrate: pressure and temperature perturbations.

Irreversible inhibition of butyrylcholinesterase by soman was studied in the presence of the substrate (o-nitrophenyl butyrate). Inhibition was found of the competitive complexing type. Study at different temperatures and pressures showed that the behavior of the enzyme differs from that of the inhibitor-free enzyme. In the absence of inhibitor, enzyme kinetics displayed a non-linear temperature dependence with a break at 21 degrees C. In the presence of a non-inhibitor structural analog of soman (pinacolyl dimethylphosphinate and methyl dimethylphosphinate), the Arrhenius plot break is slightly shifted (18 degrees C). On the other hand, in the presence of soman this break is abolished. The pressure-dependence of the substrate hydrolysis revealed also differences between the native enzyme and the enzyme in the presence of soman: the sign and magnitude of the apparent activation volume (delta V not equal to) were different for the two reactions. Beyond 300 bar, in the presence of soman, a plateau (delta V not equal to approx. 0) was observed over a large pressure range depending on temperature. Such a behavior with respect to temperature and pressure can reflect a soman-induced enzyme conformational state. Thus, temperature and pressure perturbations of the kinetics allow to complete the inhibition scheme of butyrylcholinesterase by soman. Our data suggest that upon soman binding, the enzyme undergoes a long-lived soman-induced-fit conformational change preceding the phosphonylation step. However, an alternative hypothesis according to which the enzyme processes a secondary soman-binding site cannot be ruled out.     

High-pressure stopped-flow spectrometry at low temperatures

A stopped-flow instrument operating over temperature and pressure ranges of +30 to -20 degrees C and 10(-3) to 2 kbar , respectively, is described. The system has been designed so that it can be easily interfaced with many commercially available spectrophotometers of fast response time, with the aid of quartz fiber optics. The materials used for the construction are inert, metal free and the apparatus has proven to be leak free at temperatures as low as -20 degrees C under a pressure of 2 kbar . The performance of the instrument was tested by measuring the rate of reduction of cytochrome c with sodium dithionite and the 2,6-dichloroindophenol/ascorbate reaction. The dead time of the system has been evaluated to be 20, 50, and congruent to 100 ms in water at 20 degrees C, in 40% ethylene glycol/water, and at 20 degrees C and -15 degrees C, respectively. These values are rather pressure independent up to 2 kbar . Application of the bomb was demonstrated using the cytochrome c peroxidase/ethyl peroxide reaction. This process occurred in two phases and an increase in pressure decreased the rates of reactions indicating two positive volumes of activation (delta V not equal to app (fast) = 9.2 +/- 1.5 ml X mol-1; delta V not equal to app (slow) = 14 +/- 1.5 ml X mol-1, temperature 2 degrees C). The data suggest that the fast reaction could involve a hydrophobic bond, whereas the slow process could be associated with a stereochemical change of the protein. The problem of temperature equilibrium for high-pressure experiments is also discussed.     

Effect of NaCl addition on nanosecond O2 escaping reaction of myoglobin: evidences for the transition of myoglobin dynamic structure at 20 degrees C.

We studied the nanosecond (ns) geminate O2 escape reaction from the protein interior of myoglobin (Mb) to the solvent phase in the temperature range of 5-40 degrees C containing 0-0.1 M NaCl. In the flash photolysis experiments, we found that both the rate constant, kout, and its Arrhenius plot changed upon the variation of the NaCl concentration. In particular, it was noteworthy that the Arrhenius plot of kout dramatically changed in its slope, keeping the break at 20 degrees C, upon the addition of NaCl, indicating that the thermodynamic parameters such as an enthalpy of activation (delta H not equal to) and an entropy of activation (delta S not equal to) are different between above and below 20 degrees C, and that they are further altered upon the NaCl addition to the sample solution. From these results, we suggested that the Mb dynamic structure in the ns geminate O2 escape reaction is sensitively regulated by the interaction of the protein surface and the salt. The present study also showed that an inconsistency of the Arrhenius plot of kout between Chatfield et al. ((1990) J. Am. Chem. Soc. 112, 4680-4687) and us ((1990) J. Biol. Chem. 265, 18823-18828) is probably due to the difference in the solution condition.     

Dissociation and aggregation of lactic dehydrogenase by high hydrostatic pressure

As shown by earlier experiments high hydrostatic pressure affects the catalytic function of lactic dehydrogenase from rabbit muscle. In the presence of substrates denaturation occurs, whereas in the absence of substrates and --SH-protecting reagents oxidation of sulfhydryl groups takes place [Schmid, G., Lüdemann, H.-D. & Jaenicke, R. (1975) Biophys. Chem. 3, 90--98; (1978) Eur. J. Biochem. 86, 219--224]. Avoiding oxidation effects by reducing conditions in the solvent medium and by chelation of heavy metal ions, the remaining high-pressure effects consist of dissociation of the native quaternary structure into subunits followed by aggregation. Both reactions are influenced by temperature and enzyme concentration. Short incubation (less than or equal to 10 min) at pH 6.0--8.5 and pressures of 0.3--1.0 kbar causes dissociation which is reversed at normal pressure. At 5 degrees C the activation volume is found to be delta V not equal to = -62 +/- 3cm3 . mol-1. Above 1.2 kbar irreversible aggregation takes place; the reaction is favoured by low temperature and decreased pH. The activation volume for the aggregation process at 5 degress C is delta V not equal to = -97 +/- 3cm3 . mol-1. The results may be described by a reaction sequence comprisign pressure-induced dissociation of the native enzyme into its subunits followed by subunit aggregation to form inactive high-molecular-weight particles.     

Pressure effects on catalysis by alkaline phosphatase reconstituted in lipid bilayers

Bovine intestinal alkaline phosphatase (EC 3.1.3.1) was reconstituted into lipid bilayers by a dilution method using n-octylglucopyranoside. From the kinetic measurements at various pressures, the volume of activation (delta V not equal to) and volume change in substrate binding (delta V) were estimated for free and reconstituted ALP. The delta V not equal to and delta V values for free ALP and reconstituted ALP in the gel state liposome showed opposite tendencies (-23 ml . mol-1 [delta V not equal to], 35 ml . mol-1 [delta V] for free ALP and 27 ml . mol-1 [delta V not equal to], -36 ml . mol-1 [delta V] for reconstituted ALP, respectively), which suggest both strong desolvation effect of enzyme molecule by the surrounding lipids and drastic conformational change of the enzyme molecule by the reconstitution into liposomes.     

Pressure dependence of fluorescence quenching reactions in proteins

The effect of hydrostatic pressure (0-2.6 kbar) on the acrylamide quenching of the fluorescence of indole derivatives and several single-tryptophan-containing proteins has been studied using phase fluorometry at 25 degrees C. For the model system, N-acetyl-L-tryptophanamide in water, there is essentially no pressure dependence of the quenching rate constant, kappa q. For the internal Trp residue of ribonuclease T1 and cod parvalbumin, there also is essentially no pressure dependence of the apparent kappa q at low pressure. Thus, the activation volume, delta V not equal to, for these quenching processes is approximately zero. Such small delta V not equal to values are expected for diffusion-limited reactions in water at this temperature. The low, apparent delta V not equal to values for the globular proteins characterize these quenching processes as involving very small amplitude fluctuations in the protein structures. Only for the poised tetramer in equilibrium monomer equilibrium of melittin were we able to observe a significant effect of pressure on kappa q and this is due to the pressure-induced shift in the equilibrium position.     

Effect of solvent, pressure and temperature on reaction rates of the multiheme hydroxylamine oxidoreductase. Evidence for conformational change

Hydroxylamine oxidoreductase (HAO) of the ammonia-oxidizing bacterium Nitrosomonas catalyzes the oxidation: NH2OH + H2O----HNO2 + 2e- + 2 H+. The heme-like chromophore P460 is part of a site which binds substrate, extracts electrons and then passes them to the many c hemes of the enzyme. Reduction of the c hemes by hydroxylamine is biphasic with apparent first-order rate constants k1 and k2. CO binds to ferrous P460 with apparent first-order rate constants, k1,CO. In this work we have measured the binding of CO to ferrous P460 of hydroxylamine oxidoreductase and the reduction by substrate of some of the 24 c hemes of the ferric enzyme. These reactions have been studied in water and 40% ethylene glycol, at temperatures ranging from -15 degrees C to 20.7 degrees C and at hydrostatic pressures ranging over 0.1-80 MPa. From the measurements, thermodynamic parameters delta V+ (activation volume), delta G+, delta H+, and delta S+ have been calculated. CO binding. Binding of CO to ferrous P460 was similar to the binding of CO to ferrous horseradish peroxidase. The change of solvent had only a limited effect on delta V+ (-30 ml.mol-1), delta G+, delta H+ or delta S+ and did not cause an inflection in the Arrhenius plot or downward displacement of the linear relationship between ln k1,CO and P at a critical temperature. Binding was exothermic at high temperatures. The response of the binding of CO to solvent, temperature and pressure suggested that the CO binding site had little access to solvent and was not susceptible to change in protein conformation. Fast phase of reduction of c hemes. Changing the solvent from water to 40% ethylene glycol resulted in a decrease from 90% to 50% in the relative number of c hemes reduced during the fast phase, an increase in activation volume from -3.6 ml.mol-1 to 57 ml.mol-1 and changes in other thermodynamic parameters. The activation volume increased with decreasing temperature. The Arrhenius plot had a downward inflection at about 0 degrees C and, in water or ethylene glycol, the linear dependence of ln k1 on P was displaced downwards as the temperature changed from 3.5 degrees C to -15 degrees C. Slow phase of reduction of c hemes. Changing the solvent from water to 40% ethylene glycol resulted in an increase in the relative number of c hemes reduced during the slow phase from 10% to 50%. The activation volume, which was not measurable in water because of the low absorbance change, was -30 ml.mol-1 in ethylene glycol. The activation volume increased with increasing temperature.(ABSTRACT TRUNCATED AT 400 WORDS)     

Preliminary studies on quinoprotein glucose dehydrogenase under extreme conditions of temperature and pressure.

The kinetics of the reduction of the quinoprotein glucose dehydrogenase by substrate were studied as a function of 3 parameters: pressure (1-1000 bar), temperature (down to -25 degrees C) and solvent (water and 40% dimethyl sulfoxide, DMSO) using a high-pressure low-temperature stopped-flow apparatus. A 2-step formation of the reduced enzyme by its substrate (xylose), was observed. A rapid equilibrium described by the constant K1 was followed by a slower process described by the constants k2 and k-2. By using the transition state theory, the thermodynamic quantities delta V (activation volumes) were determined for these various kinetics constants under different experimental conditions. The results are discussed in terms of conformational change and solvation effect on the protein shell, and compared with results obtained for other systems as the 2-step formation of horseradish peroxidase compound I.     

Effects of magnesium sulfate on an unfolding step of human cyanomet myoglobin.

The effects of magnesium sulfate (MgSO4) on an unfolding step of human cyanomet myoglobin (Mb) were examined for wild-type and three L-->A mutant Mbs. The unfolding was induced at acidic pH (3.6-4.5) with various concentrations of MgSO4 (0-2 M). The monophasic process was monitored by visible absorption spectroscopy. We observed quite nonlinear delta G not equal to-[MgSO4] relations for all the Mbs. delta G not equal to-[MgCl2] relations were also determined for a comparative study. Thermodynamic evaluation of the results indicated that an upward reflection of delta G not equal to-[MgSO4] relations in high [MgSO4] is caused by the strong Hofmeister effect of the salt. Results obtained for three mutants (L29A, L72A, and L104A) at pH 4.0 and 4.5 were consistent with our previous observation that the structure of the transition state is determined by the stability of Mb cores in the balance with the pH conditions of unfolding (T. Konno and I. Morishima. 1993. Biochim. Biophys. Acta. 1162:93-98).     

Effect of PEEP on the mechanics of the respiratory system in ARDS patients.

In patients with adult respiratory distress syndrome (ARDS) we studied the effect of positive end-expiratory pressure (PEEP) on respiratory mechanics. We used the technique of rapid airway occlusion during constant flow (V) inflation to partition the total respiratory system resistance (Rrs) into the interrupter resistance (Rint,rs) and the additional resistance (delta Rrs) due to viscoelastic pressure dissipations and time constant inequalities. We also measured static (Est,rs) and dynamic (Edyn,rs) elastance of the respiratory system. The procedure was carried out in nine ARDS patients at different inspiratory V and inflation volumes (delta V) at PEEP of 0, 5, 10, and 15 cmH2O. We found that during baseline ventilation (delta V = 0.7 liter and V = 1 l/s), Est,rs, Edyn,rs, and Rint,rs did not change significantly with PEEP, whereas delta Rrs and Rrs increased significantly only with PEEP of 15 cmH2O. The increase of delta Rrs and Rrs with PEEP was positively correlated with the concomitant changes in end-expiratory lung volume (P < 0.001). At all levels of PEEP, under iso-delta V conditions, delta Rrs decreased with increasing V, whereas at a fixed V, delta Rrs increased with increasing delta V. A four-parameter model of the respiratory system failed to fully describe respiratory dynamics in the ARDS patients, probably due to nonlinearities.     

NMR study of the alkaline isomerization of ferricytochrome c

The pH-induced isomerization of horse heart cytochrome c has been studied by 1H NMR. We find that the transition occurring in D2O with a pKa measured as 9.5 +/- 0.1 is from the native species to a mixture of two basic forms which have very similar NMR spectra. The heme methyl peaks of these two forms have been assigned by 2D exchange NMR. The forward rate constant (native to alkaline cytochrome c) has a value of 4.0 +/- 0.6 s-1 at 27 degrees C and is independent of pH; the reverse rate constant is pH-dependent. The activation parameters are delta H not equal to = 12.8 +/- 0.8 kcal.mol1, delta S not equal to = -12.9 +/- 2.0 e.u. for the forward reaction and delta H not equal to = 6.0 +/- 0.3 kcal.mol-1, delta S not equal to = -35.1 +/- 1.3 e.u. for the reverse reaction (pH* = 9.28). delta H degree and delta S degree for the isomerization are 6.7 +/- 0.6 kcal.mol-1 and 21.9 +/- 1.0 e.u., respectively.     

Conformational plasticity of butyrylcholinesterase as revealed by high pressure experiments

The ligand binding and kinetic behaviour of butyrylcholinesterase (EC 3.1.1.8, acylcholine acylhydrolase) from human plasma was studied at 35 degrees C under high hydrostatic pressure. The binding of phenyltrimethylammonium was studied by affinity electrophoresis at various pressures ranging from 10(-3) to 2 kbar. The kinetics of enzyme carbamylation with N-methyl(7-dimethylcarbamoxy)quinolinium iodide was studied in single-turnover conditions up to 1.2 kbar using a high-pressure stopped-flow fluorimeter. Experiments were carried out in different media: 1 mM Tris-HCl (pH 8) with water, water containing 0.1 M lithium chloride and deuterium oxide as solvents. The volume changes (delta V and delta V++) associated with each process were determined from the pressure-dependence of the binding and kinetic constants. Kinetic data show that the binding of substrate to the enzyme leads to a pressure-sensitive enzyme conformational state which cannot accomplish the catalytic act. The pressure-induced inhibitory effect is highly cooperative; it depends on both the nature (charged or neutral) and the concentration of the substrate. Also, large solvent effects indicate that enzyme sensitivity to pressure depends on the solvent structure. This findings suggests that the substrate-dependent pressure effect is modulated by the solvation state of the enzyme.