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.     

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.     

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.     

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)     

Kinetics of pressure-induced inactivation of bovine liver glutamate dehydrogenase

Kinetics of pressure-induced denaturation of bovine liver glutamate dehydrogenase (EC 1.4.1.3) were investigated in the pressure range 1.8-2.8 kbar by observing the residual activity after the pressure-release and the scattered light intensity during the incubation at high pressure. The residual activity decreased exponentially with the incubation time, whereas the scattered light intensity showed a bimodal profile indicating parallel aggregation and dissociation reactions. The latter suggested that two kinds of aggregates were formed during the incubation under pressure. The observed first-order rate constant for the inactivation, k obs, showed a minimum around 30 degrees C. These experimental results were interpreted in terms of the following reaction scheme; (formula; see text) where N represents the enzyme entity with native structure, D1 the partially denatured intermediate, D2 the irreversibly denatured state, and A1 and A2 the two kinds of aggregates, one of which (A1) is reversibly formed at an early stage of the incubation under high pressure. The apparent activation volume for the inactivation reaction was estimated to be delta V*app = -113 +/- 5 cm3 X mol-1 from the pressure dependence of k obs. The effect of coenzyme, NAD+, on the pressure-induced inactivation was also studied. The inactivation was retarded by the presence of the coenzyme, whereas the apparent activation volume for the holoenzyme (delta V*app = -104 +/- 2 cm3 X mol-1) did not differ significantly from that for the apoenzyme.     

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.     

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.     

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.     

Effects of pressure on the kinetics of capture by yeast alcohol dehydrogenase.

High pressure causes biphasic effects on the oxidation of benzyl alcohol by yeast alcohol dehydrogenase as expressed in the kinetic parameter V/K which measures substrate capture. Moderate pressure increases the rate of capture of benzyl alcohol by activating the hydride transfer step. This means that the transition state for hydride transfer has a smaller volume than the free alcohol plus the capturing form of enzyme, with a DeltaV of -39 +/- 1 mL/mol, a value that is relatively large. This is the first physical property of an enzymatic transition state thus characterized, and it offers new possibilities for structure-activity analyses. Pressures of >1.5 kbar decrease the rate of capture of benzyl alcohol by favoring a conformation of the enzyme which binds nicotinamide adenine dinucleotide (NAD+) less tightly. This means that the ground state for tight binding, E-NAD+, has a larger volume than the collision complex, E-NAD+, with a DeltaV of 73 +/- 2 mL/mol. The equilibrium constant of the conformational change Keq is 75 +/- 13 at 1 atm. The effects of pressure on the capture of NAD+ have no activation phase because the conformational change is now being expressed kinetically instead of thermodynamically, together with but in opposition to hydride transfer, causing the effects to cancel. For yeast alcohol dehydrogenase, this conformational change had not been detected previously, but similar conformational changes have been found by spectroscopic means in other dehydrogenases, and some of them are also sensitive to pressure. The opposite signs for the volume change of tighter binding and hydride transfer run contrary to Pauling's hypothesis that substrates are bound more tightly in the transition state than in the Michaelian reactant state.     

Kinetic evidence for thermally induced conformational change of butyrylcholinesterase

The effect of temperature on the kinetics of human plasma butyrylcholinesterase-catalyzed reactions was studied. The Arrhenius plot of o-nitrophenylbutyrate hydrolysis presents a break at 21 degrees C. However, nucleophilic competition data indicate that there is no change in the rate-limiting step of the overall reaction. In addition, the temperature dependence of the bimolecular rate constant of enzyme carbamylation shows a break at 18 degrees C. These results argue for the existence of thermally induced conformational active states of the enzyme tetramer. It is suggested that the effects of this transition on kinetics arise at the acylation 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.     

A rapid reaction study of anthranilate hydroxylase. Evidence for a catalytically important conformational change during slow initial turnover with anthranilate

Rapid reaction kinetics of the flavoprotein anthranilate hydroxylase from Trichosporon cutaneum were examined for reactions involving anthranilate, the native substrate. As was reported earlier for the nonhydroxylated substrate analogue, salicylate, some reactions in the first turnover with anthranilate occur slower than those in subsequent turnovers (Powlowski, J., Massey, V., and Ballou, D. P. (1989) J. Biol. Chem. 264, 5606-5612). Evidence is presented for slow conformational changes that occur both on binding of the aromatic ligand and on reduction of the enzyme. These changes are apparently important for rapid anthranilate binding to occur in turnovers subsequent to the first. Moreover, bound anthranilate is required for rapid reduction of enzyme-bound FAD by NADPH. Studies to probe the accessibility of reagents to modified flavins that had been incorporated into the apoenzyme indicate that anthranilate binding causes a conformational change in the protein, allowing increased access to the benzene ring moiety of the flavin. An unusual isotope effect with (R)-NADPD (4(R)-2H] NADPH) is observed on Kd rather than on kred, which is consistent with a model involving slow interconversion of enzyme-substrate complexes before productive binding of NADPH and reduction of the enzyme flavin.     

Differential effect of pressure and temperature on the catalytic behaviour of wild-type human butyrylcholinesterase and its D70G mutant.

The combined action of temperature (10-35 degrees C) and pressure (0. 001-2 kbar) on the catalytic activity of wild-type human butyrylcholinesterase (BuChE) and its D70G mutant was investigated at pH 7.0 using butyrylthiocholine as the substrate. The residue D70, located at the mouth of the active site gorge, is an essential component of the peripheral substrate binding site of BuChE. Results showed a break in Arrhenius plots of wild-type BuChE (at Tt approximately 22 degrees C) whatever the pressure (dTt/dP = 1.6 +/- 1.5 degrees C.kbar-1), whereas no break was observed in Arrhenius plots of the D70G mutant. These results suggested a temperature-induced conformational change of the wild-type BuChE which did not occur for the D70G mutant. For the wild-type BuChE, at around a pressure of 1 kbar, an intermediate state, whose affinity for substrate was increased, appeared. This intermediate state was not seen for the mutant enzyme. The wild-type BuChE remained active up to a pressure of 2 kbar whatever the temperature, whereas the D70G mutant was found to be more sensitive to pressure inactivation (at pressures higher than 1.5 kbar the mutant enzyme lost its activity at temperatures lower than 25 degrees C). The results indicate that the residue D70 controls the conformational plasticity of the active site gorge of BuChE, and is involved in regulation of the catalytic activity as a function of temperature.     

A substrate-induced conformation change in the reaction of alkaline phosphatase from Escherichia coli

1. Benzyl phosphonates were prepared and their potentialities as chromophoric reagents for the exploration of the substrate-binding site of Escherichia coli alkaline phosphatase were investigated. 4-Nitrobenzylphosphonate is a competitive inhibitor of the enzyme. 2-Hydroxy-5-nitrobenzylphosphonate changes its spectrum on binding to the enzyme. This spectral change is reversed when the phosphonate is displaced from the enzyme by substrate. 2. The kinetics of the reaction of 2-hydroxy-5-nitrophenylphosphonate were studied by the stopped-flow and the temperature-jump techniques. It was found that the combination of the phosphonate with the enzyme occurred in two successive and reversible steps: enzyme-phosphonate complex-formation followed by rearrangement of the complex. The spectral change is associated with the rearrangement. At pH8 in 1m-sodium chloride at 22 degrees the rate constant is 167sec.(-1) for the rearrangement of the initially formed binary complex and is 18sec.(-1) for the reverse process. 3. It has previously been proposed that the reactions of phosphatase with its substrates include a distinct step between enzyme-substrate combination and chemical catalysis. The rate constant involved could be predicted but not measured from experiments with substrates. The value for the rate constant measured from the rate of the enzyme-phosphonate rearrangement is in excellent agreement with the predicted value. A model for the reaction mechanism is proposed that includes a conformation change in response to phosphate ester binding before phosphate transfer from substrate to enzyme.     

Effects of cell volume regulating osmolytes on glycerol 3-phosphate binding to triosephosphate isomerase

During cell volume regulation, intracellular concentration changes occur in both inorganic and organic osmolytes in order to balance the extracellular osmotic stress and maintain cell volume homeostasis. Generally, salt and urea increase the Km's of enzymes and trimethylamine N-oxide (TMAO) counteracts these effects by decreasing Km's. The hypothesis to account for these effects is that urea and salt shift the native state ensemble of the enzyme toward conformers that are substrate-binding incompetent (BI), while TMAO shifts the ensemble toward binding competent (BC) species. Km's are often complex assemblies of rate constants involving several elementary steps in catalysis, so to better understand osmolyte effects we have focused on a single elementary event, substrate binding. We test the conformational shift hypothesis by evaluating the effects of salt, urea, and TMAO on the mechanism of binding glycerol 3-phosphate, a substrate analogue, to yeast triosephosphate isomerase. Temperature-jump kinetic measurements promote a mechanism consistent with osmolyte-induced shifts in the [BI]/[BC] ratio of enzyme conformers. Importantly, salt significantly affects the binding constant through its effect on the activity coefficients of substrate, enzyme, and enzyme-substrate complex, and it is likely that TMAO and urea affect activity coefficients as well. Results indicate that the conformational shift hypothesis alone does not account for the effects of osmolytes on Km's.     

High-pressure investigations of cytochrome P-450 spin and substrate binding equilibria

The effects of high pressure (1-2000 bar) on the spin state and substrate binding equilibria in cytochrome P-450 have been determined. The high-spin (S = 5/2) to low spin (S = 1/2) transition of the ferric hemoprotein was monitored by uv-visible spectroscopy at various substrate concentrations. Increasing hydrostatic pressure on a sample of substrate-bound cytochrome P-450 resulted in a decrease in the high-spin fraction as monitored by a Soret maxima at 391 nm and an increase in the low-spin 417-nm region of the spectrum. These pressure-induced optical changes were totally reversible for all pressures below 800 bar and were found to correspond to simple substrate dissociation from the enzyme. High levels of the normally metabolized substrate, d-camphor, corresponding to a 99.9% saturation of the hemoprotein active site (50 mM Tris-Cl, 100 mM KCl, pH 7.2) completely prevented the pressure-induced high-spin to low-spin transition that is observed at less than saturating substrate concentrations. A gradual increase in the formation of the inactive P-420 form of the cytochrome was noted if the pressure of the sample was increased above 800 bar. These pressure-linked spectral changes were used to determine the microscopic volume change accompanying substrate binding, which was found to be -47.0 +/- 2 ml/mol (pH 7.2) which represents a substantial change for a ligand dissociation reaction. The observed volume change for camphor binding decreases to -30.6 +/- 2 ml/mol at pH 6.0, suggesting the involvement of a linked proton equilibrium. Various substrate analogs of camphor induce varying degrees of low-spin to high-spin shift upon binding to ferric cytochrome P-450 (3). The volume changes for the dissociation of these substrates were very similar to those obtained with camphor. The conformational changes associated with a shift from high- to low-spin ferric iron appear to be small in comparison to the overall macroscopic changes in volume accompanying substrate binding to the enzyme.     

The effects of pressure on yeast cytochrome c peroxidase

The effects of pressure on cytochrome c peroxidase [CcP(FeIII)], its cyano derivative (CcP X CN) and its enzyme-substrate complex (ES) have been studied. The effects of pressure on the binding of the substrate analog porphyrin cytochrome c (porphyrin c) to CcP X CN and ES have also been studied. High pressure causes CcP(FeIII) to undergo a high-spin to low-spin transition but has no detectable effect on either CcP X CN, which is already low spin, or on ES. The low-spin CcP(FeIII) structure at pressure is similar to the low-spin form at low temperature and the low-spin form of horseradish peroxidase at high pressure. delta V degree associated with the spin equilibrium is about 30 ml/mol and is independent of temperature. delta G degree is small, 4.7 kJ/mol at 0 degree C, while delta H degree is 14.2 kJ/mol at 1 bar (100 kPa). Pressure has no detectable effect on the binding equilibria of mixtures of CcP X CN plus porphyrin c or ES plus porphyrin c. This indicates that the interaction of CcP and porphyrin c results in little or no volume change; the same is true in the case of cytochrome c oxidase and porphyrin c.     

Thermodynamic arguments for temperature-induced cryptic conformational change of human plasma cholinesterase

The temperature and pressure dependence of the kinetics of the hydrolysis of o-nitrophenylbutyrate by human plasma tetrameric form cholinesterase (EC 3.1.1.8) was studied. The study was carried out on the one hand at atmospheric pressure by spectrophotometry at various temperatures ranging from 0 to 40 degrees C and, on the other hand by high-pressure stopped-flow spectrophotometry at 3.5, 25 and 35 degrees C in the pressure range 10(-3) to 2 kbar. The Arrhenius plot showed a break at 21 +/- 1 degrees C. Kinetic parameters, activation parameters and volume changes are reported. Discontinuities in the thermodynamic quantities obtained from temperature and pressure (up to 0.8 kbar) dependence of hydrolysis rates are discussed; they have been interpreted as the result of a temperature-induced cryptic conformational change of the enzyme at around 20 degrees C. Beyond 1 kbar the kinetics exhibited several complexities: curvature of the progress curves and high positive or negative activation volume changes depending on temperature and substrate concentration. These complex interacting effects between temperature, pressure and substrate concentration are discussed.     

Crystal structure of substrate complexes of methylmalonyl-CoA mutase.

X-ray crystal structures of methylmalonyl-CoA mutase in complexes with substrate methylmalonyl-CoA and inhibitors 2-carboxypropyl-CoA and 3-carboxypropyl-CoA (substrate and product analogues) show that the enzyme-substrate interactions change little during the course of the rearrangement reaction, in contrast to the large conformational change on substrate binding. The substrate complex shows a 5'-deoxyadenine molecule in the active site, bound weakly and not attached to the cobalt atom of coenzyme B12, rotated and shifted from its position in the substrate-free adenosylcobalamin complex. The position of Tyralpha89 close to the substrate explains the stereochemical selectivity of the enzyme for (2R)-methylmalonyl-CoA.