The standard Gibbs free energy change of hydrolysis of alpha-D-ribose 1-phosphate

The standard Gibbs free energy change of hydrolysis of α-d-ribose 1-phosphate has been measured at pH 7.0, ionic strength 0.1 m, and 25 °C by combining the corresponding values of the two following reactions: adenosine + H₂O ág adenine + ribose (ΔG^0′ = ?2.3 ± 0.1 kcal/mol), catalyzed by adenosine nucleosidase, and ribose 1-phosphate + adenine ág adenosine + P_i (ΔG^0′ = ?3.1 ± 0.1 kcal/mol), catalyzed by adenosine phosphorylase. The standard Gibbs free energy changes were calculated for both reactions from the equilibrium constant. A value of -5.4 ± 0.15 kcal/mol, comparable to that of other hemiacetal phosphoric esters, was obtained for the hydrolysis of ribose 1-phosphate.     

Magnesium-dependent ATPase activity and cooperativity of magnesium chelatase from Synechocystis sp. PCC6803

The first committed step in chlorophyll biosynthesis is catalyzed by magnesium chelatase, a complex enzyme with at least three substrates, cooperative Mg(2+) activation, and free energy coupling between ATP hydrolysis and metal-ion chelation. A detailed functional study of the behavior of the intact magnesium chelatase has been performed, including characterization of magnesium cooperativity and the stoichiometry of ATP consumption in relation to the magnesium porphyrin produced. It is demonstrated that, in vitro, this catalyzed reaction requires hydrolysis of approximately 15 MgATP(2-) and that the chelation partial reaction is energetically unfavorable, under our assay conditions, with a DeltaG degrees ' of 25-33 kJ mol(-1). Given the likely metabolite concentrations in vivo, this results in the chelatase reaction operating far from equilibrium. We have also determined the steady-state kinetic behavior of the intact enzyme and have compared the kinetic parameters obtained with those observed for the partial reactions of individual subunits. K(DIX) (where D(IX) represents deuteroporphyrin IX) is estimated to be 3.20 microm, and K(MgATP)(2-) is 0.45 mm. k(cat) for chelation is estimated to be 0.8 min(-1), suggesting that the ATP hydrolysis catalyzed by the isolated ChlI subunit is substantially slower in the intact chelatase. The magnesium-rich form of the chelatase is a more effective catalyst of the chelation reaction; magnesium activation of the chelatase increases V, as well as the specificity constant for the reaction of MgATP(2-) and D(IX), possibly as a result of a magnesium-triggered conformational change.     

Synthesis of triglycerides of phenylbutyric acids by lipase-catalyzed glycerolysis in a solvent-free system

Triphenylbutyrin, a triglyceride prodrug of 4-phenylbutyric acid, has potential for use in drug delivery systems. Immobilized Candida antarctica lipase B catalyzed the synthesis of triphenylbutyrin by glycerolysis of 4-phenylbutyrate with glycerol in a solvent-free system. The use of 4-phenylbutyate with a leaving alcohol moiety in the acyl-transfer reaction can shift the equilibrium of the reaction toward the synthesis of triphenylbutyrin, since the alcohol by-product can be removed by vacuum. A 97% yield of triphenylbutyrin was achieved in a solvent-free system at a reaction temperature of 75 °C by combination of molecular sieves to limit the hydrolysis side reaction and a vacuum system to shift the reaction equilibrium in a solvent-free system.     

Coated-wall microreactor for continuous biocatalytic transformations using immobilized enzymes

Microstructured flow reactors are emerging tools for biocatalytic process development. A compelling design is that of the coated-wall reactor where enzyme is present as a surface layer attached to microchannel walls. However, preparation of a highly active wall biocatalyst remains a problem. Here, a stainless steel microreactor was developed where covalent immobilization of the enzyme in multiple linear flow channels of the reaction plate was supported by a macroporous wash-coat layer of gamma-aluminum oxide. Using surface functionalization with aminopropyl triethoxysilane followed by activation with glutardialdehyde, the thermophilic beta-glycosidase CelB from Pyrococcus furiosus was bound with retention of half of the specific activity of the free enzyme (800 U/mg), yielding a high catalyst loading of about 500 U/mL. This microreactor was employed for the continuous hydrolysis of lactose (100 mM) at 80 degrees C, providing a space-time yield of 500 mg glucose/(mL h) at a stable conversion of > or =70%. The immobilized enzyme displayed a half-life of 15 days under the operational conditions. Due to the absence of hydrophobic solute-material interactions, which limit the scope of microstructures fabricated from poly(dimethylsiloxane) for biocatalytic applications, the new microreactor was fully compatible with the alternate enzyme substrate 2-nitro-phenyl-beta-D-galactoside and the 2-nitro-phenol product resulting from its hydrolysis catalyzed by CelB.     

Thermodynamic parameters monitoring the equilibrium shift of enzyme-catalyzed hydrolysis/synthesis reactions in favor of synthesis in mixtures of water and organic solvent

The main strategy developed to shift the equilibrium state of a hydrolase-catalyzed hydrolysis/synthesis reaction consists in reducing water activity by addition of organic solvents in the reaction medium. We have used several mixtures of water and 1,4-butanediol, ranging from pure water to pure 1,4-butanediol, to study the hydrolysis/synthesis reaction of the N-Cbz-L-tryptophanyl-glycineamide dipeptide, catalyzed by alpha-chymotrypsin. In the presence of 1,4-butanediol, alpha-chymotrypsin also catalyzed the esterification reaction between this diol and N-Cbz-L-tryptophan; this ester hydrolysis/synthesis reaction has thus also been examined. The dipeptide and ester equilibrium concentrations increase when the water content of the reaction medium is decreased. Using our experimental data, we have determined the equilibrium constants of the hydrolysis/synthesis equilibria involving the nonionized forms of the protected amino acids, the estimated values of which are Ksp = 8 10(5) for the dipeptide and Kse = 78 for the ester respectively. They are true thermodynamic equilibrium constants, each related to a single, well-defined reaction equilibrium and with water activity being taken into account. If an organic solvent is added to the reaction medium these equilibria can be shifted towards synthesis by decreasing the water activity but also by modifying the ionization/neutralization equilibrium constant of the ionizable groups. These two effects depend both on the water content and on the nature of the organic solvent used, and, in particular, on its dielectric constant. Because of the importance of this parameter in our study, we discuss using it as an indicator to select an appropriate organic solvent to perform an enzyme-catalyzed synthesis.     

Reaction pathways for Zn(II)-catalyzed carboxylic acid esters hydrolysis

Reaction pathways have been investigated by quantum-mechanical procedures on gas phase models for the hydrolysis of methyl acetate catalyzed by a monohydroxo-Zn(II) complex formed by a phenanthroline-containing polyamine macrocycle. Based on consideration of energy barrier heights, the hydrolysis process is predicted to be bimolecular, consistently with kinetic data obtained for the hydrolysis of p-nitrophenyl acetate promoted by the modelled catalyst. Differences with respect to results of theoretical studies on the more extensively investigated hydrolysis processes catalyzed by the OH⁻ anion are discussed and appear to be mostly due to the presence of the metal centre close to the OH function in the system now investigated. The pervasive presence and path-controlling role of hydrogen bonds are also discussed.     

A Desolvated Solid–Solid Interface for a High‐Capacitance Electric Double Layer

Understanding the electric double layer is essential for achieving efficient electrochemical energy storage technologies. A conventional solid–liquid electrode interface suffers from serious self‐discharge and a narrow voltage window, which makes the development of a solid–solid interface imperative. However, an in‐depth understanding of the electric double layer with a solid–solid interface is lacking. Here, a solid–solid interfacial electric double layer is proposed with excellent electrochemical performance. The solid layer is constructed by the electrochemical decomposition of lithium difluoro(oxalate)borate, which provides a desolvated environment for the establishment of a electric double layer. This makes a stronger interaction between the electrode surface and the ions. Based on this unique property, it is found that the solid–solid interfacial electric double layer has an increased capacitance, which suggests a way to develop high‐energy electrochemical capacitors.     

Multiple ion-dependent and substrate-dependent Na+/K+-ATPase conformational states. Transient and steady-state kinetic studies

The hydrolysis of beta-(2-furyl)acryloyl phosphate (FAP), catalyzed by the Na+/K+-ATPase, is faster than the catalyzed hydrolysis of ATP. This is due to catalyzed hydrolysis of the pseudosubstrate by K+-dependent states of the enzyme, thus bypassing the Na+-dependent enzyme states that are required and are rate limiting in ATP hydrolysis. Unlike ATP, FAP is a positive effector of the E2 state. A study of FAP hydrolysis permits a detailed analysis of later steps in the overall ion translocation-ATP hydrolysis pathway. During the steady state of FAP hydrolysis in the presence of K+, substantial phosphoryl-enzyme is formed, as is indicated by the covalent incorporation of 32P from [32P]FAP. A comparison of the phosphoryl-enzyme yield with the rate of overall hydrolysis reveals that at 25 degrees C the phosphoryl-enzyme formed is all kinetically competent. Both the yield of phosphoryl-enzyme and the rate of overall hydrolysis of FAP are [K+] dependent. The transition E1 in equilibrium E2 is also [K+] dependent, but the rate of transition is differently affected by [K+] than are the above-mentioned two processes. Two distinct roles for K+ are indicated, as an effector of the E1-E2 equilibrium and as a "catalyst" in the hydrolysis of the E2-P. In contrast to the results at 25 degrees C, a virtually stoichiometric yield of phosphoryl-enzyme occurs at 0 degree C in the presence of Na+ and the absence of K+. At lower concentrations of K+ and in the presence of Na+, the hydrolysis of FAP at 0 degree C proceeds substantially through the E1-E2 pathway characteristic of ATP hydrolysis. The selectivity of FAP for the E2-K+-dependent pathway is due to the thermal inactivation of E1 at 25 degrees C in the absence of ATP or ATP analogues, even at high concentrations of Na+. These results emphasize the existence of multiple functional "E1" and "E2" states in the overall ATPase-ion translocation pathway.     

Mechanistic study of CMP-Neu5Ac hydrolysis by α2,3-sialyltransferase from Pasteurella dagmatis

Bacterial sialyltransferases of the glycosyltransferase family GT-80 exhibit pronounced hydrolase activity toward CMP-activated sialyl donor substrates. Using in situ proton NMR, we show that hydrolysis of CMP-Neu5Ac by Pasteurella dagmatis α2,3-sialyltransferase (PdST) occurs with axial-to-equatorial inversion of the configuration at the anomeric center to release the α-Neu5Ac product. We propose a catalytic reaction through a single displacement-like mechanism where water replaces the sugar substrate as a sialyl group acceptor. PdST variants having His²⁸⁴ in the active site replaced by Asn, Asp or Tyr showed up to 10⁴-fold reduced activity, but catalyzed CMP-Neu5Ac hydrolysis with analogous inverting stereochemistry. The proposed catalytic role of His²⁸⁴ in the PdST hydrolase mechanism is to facilitate the departure of the CMP leaving group.     

Primuline Derivatives That Mimic RNA to Stimulate Hepatitis C Virus NS3 Helicase-catalyzed ATP Hydrolysis

ATP hydrolysis fuels the ability of helicases and related proteins to translocate on nucleic acids and separate base pairs. As a consequence, nucleic acid binding stimulates the rate at which a helicase catalyzes ATP hydrolysis. In this study, we searched a library of small molecule helicase inhibitors for compounds that stimulate ATP hydrolysis catalyzed by the hepatitis C virus (HCV) NS3 helicase, which is an important antiviral drug target. Two compounds were found that stimulate HCV helicase-catalyzed ATP hydrolysis, both of which are amide derivatives synthesized from the main component of the yellow dye primuline. Both compounds possess a terminal pyridine moiety, which was critical for stimulation. Analogs lacking a terminal pyridine inhibited HCV helicase catalyzed ATP hydrolysis. Unlike other HCV helicase inhibitors, the stimulatory compounds differentiate between helicases isolated from various HCV genotypes and related viruses. The compounds only stimulated ATP hydrolysis catalyzed by NS3 purified from HCV genotype 1b. They inhibited helicases from other HCV genotypes (e.g. 1a and 2a) or related flaviviruses (e.g. Dengue virus). The stimulatory compounds interacted with HCV helicase in the absence of ATP with dissociation constants of about 2 μm. Molecular modeling and site-directed mutagenesis studies suggest that the stimulatory compounds bind in the HCV helicase RNA-binding cleft near key residues Arg-393, Glu-493, and Ser-231.     

A modified theory of the potential and inhomogeneous ion distributions near charged surfaces

A new method is proposed to derive ion distributions and the electric potential near charged surfaces. The diffuse double layer is described by a proposed thermodynamic equilibrium condition between electrostatic forces and osmotic pressure. Problems related with the osmotic pressure and the integration of Coulomb forces are investigated in this paper. A numeric example is given based on erythrocyte data.     

Dynamic conformational equilibria in the physiological function of the Bombyx mori pheromone-binding protein

The Bombyx mori pheromone-binding protein (BmorPBP) undergoes a pH-dependent conformational transition from a form at basic pH, which contains an open cavity suitable for ligand binding (BmorPBP^B), to a form at pH 4.5, where this cavity is occupied by an additional helix (BmorPBP^A). This helix α7 is formed by the C-terminal dodecapeptide 131-142, which is flexibly disordered on the protein surface in BmorPBP^B and in its complex with the pheromone bombykol. Previous work showed that the ligand-binding cavity cannot accommodate both bombykol and helix α7. Here we further investigated mechanistic aspects of the physiologically crucial ejection of the ligand at lower pH values by solution NMR studies of the variant protein BmorPBP(1-128), where the C-terminal helix-forming tetradecapeptide is removed. The NMR structure of the truncated protein at pH 6.5 corresponds closely to BmorPBP^B. At pH 4.5, BmorPBP(1-128) maintains a B-type structure that is in a slow equilibrium, on the NMR chemical shift timescale, with a low-pH conformation for which a discrete set of ¹⁵N-¹H correlation peaks is NMR unobservable. The full NMR spectrum was recovered upon readjusting the pH of the protein solution to 6.5. These data reveal dual roles for the C-terminal tetradecapeptide of BmorPBP in the mechanism of reversible pheromone binding and transport, where it governs dynamic equilibria between two locally different protein conformations at acidic pH and competes with the ligand for binding to the interior cavity.     

Preparation of S-(+)-ketoprofen by coupling the enantioselective hydrolysis of ketoprofen methyl ester with the photo-oxidation of methanol

A novel method for preparation of S-(+)-ketoprofen is presented involving coupling enantioselective hydrolysis of ketoprofen methyl ester catalyzed by a surfactant-coated-lipase with the photo-oxidation of methanol in a water-saturated organic solvent. The effect of photocatalytic conversion of methanol into water and carbon dioxide on the hydrolysis of ketoprofen methyl ester and the stability of the enzyme was investigated. The photo-oxidation of methanol shifted the equilibrium of the hydrolysis toward the formation of ketoprofen, increasing the equilibrium conversion ratio and improving the enantioselectivity. Because the surfactant-coated lipase and ketoprofen methyl ester dissolved in the organic solvent and ketoprofen was absorbed on the TiO₂ photocatalyst particles, the separation procedures could be simplified and the stability of the enzyme was increased.     

Kinetics of Inhibition of Green Crab (Scylla Serrata) Alkaline Phosphatase by Sodium (2,2′-bipyridine) Oxodiperoxovanadate

Green crab (Scylla serrata) alkaline phosphatase (EC 3.1.3.1) is a metalloenzyme, which catalyzes the nonspecific hydrolysis of phosphate monoesters. The kinetics of inhibition of the enzyme by sodium (2, 2′-bipyridine) oxodiperoxovanadate, pV(bipy), has been studied. The time course of the hydrolysis of p-nitrophenyl-phosphate catalyzed by the enzyme in the presence of different pV(bipy) concentrations showed that at each pV(bipy) concentration, the rate decreased with increasing time until a straight line was approached, the straight line slopes are the same for all concentrations. The results suggest that the inhibition of the enzyme by pV(bipy) is a slow, reversible reaction with fractional remaining activity. The microscopic rate constants are determined for the reaction of inhibitor with the enzyme.     

Hydrolysis of p-nitrotrifluoroacetanilide catalyzed by water and imidazole

The hydrolyses of p-nitrotrifluoroacetanilide catalyzed by water and imidazole were examined at 70°C. The pH-rate constant profile of the hydrolysis in H₂O was examined in the pH range 0.0–11.4. The hydrolysis was independent of pH in the region from pH 1.0 to 4.5, presumably a water-catalyzed reaction. The rate constant and the D₂O solvent isotope effect for this reaction were 1.0 × 10^?4 sec^?1 and 3.7, respectively. Both natural imidazole and imidazolium cation catalyzed hydrolysis. The rate constant of the hydrolysis catalyzed by neutral imidazole was determined to be 5.4 × 10^?3M^?1 sec^?1 and the D₂O solvent isotope effect was 1.8.     

Catalysis of partial reactions of ATP synthesis by beef heart mitochondrial adenosine triphosphatase

We have found that when the ATP hydrolysis activity of beef heart mitochondrial adenosine triphosphatase (F1) is eliminated by either cold treatment or chemical modification, the enzyme attains the ability to catalyze the Pi in equilibrium ATP exchange reaction. The ATP hydrolysis activity of isolated F1 was lost upon chemical modification by phenyglyoxal, butanedione, or 7-chloro-4-nitrobenzene-2-oxa-1,3-diazole. The F1 thus chemically modified was able to catalyze an ADP-dependent Pi in equilibrium ATP exchange reaction. In addition F1 that had been cold-treated to eliminate ATP hydrolysis activity, also catalyzed the Pi in equilibrium ATP exchange reaction. The Pi in equilibrium ATP exchange catalyzed by modified F1 was shown to be totally inhibited by the F1-specific antibiotic efrapeptin. We have previously shown that isolated beef heart mitochondrial ATPase will catalyze the formation of a transition state analog of the ATP synthesis reaction (Bossard, M. J., Vik, T. A., and Schuster, S. M. (1980) J. Biol. Chem. 255, 5342-5346). While the F1-catalyzed ATP hydrolysis activity was lost rapidly upon chemical modification or cold treatment, the ability of the enzyme to produce Pi . adenosine 5'-diphosphate (chromium(III) salt) from phosphate and monodentate adenosine 5'-diphosphate (chromium(III) salt) was unimpaired. The implications of these data with regard to the mechanism of ATP synthesis are discussed.     

Allosteric properties and the association equilibria of hemocyanin from Callianassa californiensis.

The influence of magnesium ions, hydrogen ions, and oxygen on the monomer (17 S)-tetramer (39 S) equilibrium of the hemocyanin from Callianassa californiensis has been investigated. Data have been interpreted in terms of a theory integrating the allosteric equilibria and association equilibrium. Binding of oxygen and divalent cations by the hemocyanin has also been studied in terms of the theory, and the parameters in the model have been determined. The 17 S monomer, which contains six polypeptide chains, is found to be the allosteric unit which behaves as a self-contained co-operative system with allosteric properties. Both magnesium and hydrogen ions are shown to affect the association directly, whereas the effect of oxygen binding can be explained in terms of a difference in the allosteric equilibrium constant L′ in the different associated states of the protein. It is shown that whereas the effect of oxygen on the monomer-tetramer equilibrium is easily observable, the converse effect of the association equilibrium on the oxygen binding curve is at the limit of detectability.     

Influence of the Ionic Composition of Fluid Medium on Red Cell Aggregation

The effects of ionic strength and cationic valency of the fluid medium on the surface potential and dextran-induced aggregation of red blood cells (RBC's) were investigated. The zeta potential was calculated from cell mobility in a microelectrophoresis apparatus; the degree of aggregation of normal and neuraminidase-treated RBC's in dextrans (Dx 40 and Dx 80) was quantified by microscopic observation, measurement of erythrocyte sedimentation rate, and determination of low-shear viscosity. A decrease in ionic strength caused a reduction in aggregation of normal RBC's in dextrans, but had no effect on the aggregation of neuraminidase-treated RBC's. These findings reflect an increase in electrostatic repulsive force between normal RBC's by the reduction in ionic strength due to (a) a decrease in the screening of surface charge by counter-ions and (b) an increase in the thickness of the electric double layer. Divalent cations (Ca⁺⁺, Mg⁺⁺, and Ba⁺⁺) increased aggregation of normal RBC's in dextrans, but had no effect on the aggregation of neuraminidase-treated RBC's. These effects of the divalent cations are attributable to a decrease in surface potential of normal RBC's and a shrinkage of the electric double layer. It is concluded that the surface charge of RBC's plays a significant role in cell-to-cell interactions.     

Thermodynamic profiles of penicillin G hydrolysis catalyzed by wild-type and Met→Ala168 mutant penicillin acylases from Kluyvera citrophila

The Met-168 residue in penicillin acylase from Kluyvera citrophila was changed to Ala by oligonucleotide site-directed mutagenesis. The Ala-168 mutant exhibited different substrate specificity than wild-type and enhanced thermal stability. The thermodynamic profiles for penicillin G hydrolysis catalyzed by both enzymes were obtained from the temperature dependence of the steady-state kinetic parameters K_m and k_cat. The high values of enthalpy and entropy of activation determined for the binding of substrate suggest that an induced-fit-like mechanism takes place. The Met→Ala168 mutation unstabilizes the first transition-state (E··S^≠) and the enzyme-substrate complex (ES) causing a decrease in association equilibrium and specificity constants in the enzyme. However, no change is observed in the acyl-enzyme formation. It is concluded that residue 168 is involved in the enzyme conformational rearrangements caused by the interaction of the acid moiety of the substrate at the active site.     

Equilibrium shifts in enzyme reactions at high pressure

The effect of pressure on the equilibrium of a reaction was studied. Theoretical equilibrium constants and product concentrations have been calculated at elevated pressures. The theory is illustrated with an example of l-malate synthesis catalyzed by a fumarase. To study shifts in the equilibrium relatively low pressures can be applied (50–200 MPa), but our calculations show that for process optimisation much higher pressures (up to 1000 MPa) have to be used.

At these higher pressures, more stable enzymes are needed. We performed experiments with the hyperthermophilic β-glycosidase from Pyrococcus furiosus as a catalyst. Oligosaccharides were synthesized from glucose in an equilibrium reaction at pressures from 0.1 to 500 MPa. The enzyme remained active at 500 MPa. The equilibrium of the reaction was influenced by pressure and shifted towards the hydrolysis side, decreasing final oligosaccharide concentrations with increasing pressure. This pressure dependence of the final product concentration and the equilibrium constant could be described with a positive reaction volume of 2.4 mol/cm³.