Lysozyme activity in the presence of nonionic detergent micelles

The effect of a nonionic surfactant, polyoxyethylenesorbitan monolaurate (Tween 20), on the hen egg-white lysozyme catalyzed lysis of a dried cell suspension of Micrococcus lysodeikticus is analysed. A rate enhancement of up to 70% is observed in the presence of surfactant at concentrations above the critical micelle concentration. This activity increase may be explained by postulating the existence of a micelle-enzyme complex in which enzyme molecules are bound to micelles with preferential orientation of their active sites. The reaction is found to be second order with respect to substrate. A mechanism is postulated in which a substrate particle is assumed to be an energy-furnishing collision partner to the enzyme-substrate complex. This mechanism correlated data over a wide range of enzyme and substrate concentrations. Data from kinetic, ultrafiltration, ultraviolet, and fluorescence studies provide convincing evidence for the existence of a micelle-lysozyme complex. The results suggest that it is possible that immobilized enzymes mat in general be more reactive than corresponding free enzymes.     

Extraction of lysozyme and ribonuclease-a using reverse micelles: Limits to protein solubilization

Experiments are reported here on the equilibrium partitioning of lysozyme and ribonuclease-a between aqueous and reversed micellar phases comprised of an anionic surfactant, sodium di-2-ethylhexyl sulfosuccinate (AOT), in isooctane. A distinct maximum, [P](rm,max) was found for the quantity of a given protein that can be solubilized in the reverse micelle phase by the phase-transfer method. This upper limit depended upon the size of the protein, the surfactant concentration, and the aqueous phase ionic strength, and was determined by complex formation between protein and surfactant molecules to form an insoluble interfacial precipitate at high values of [P](rm). In this work, it was found to be possible to dissociate the protein-surfactant complex and recover the precipitated protein. The kinetics of protein-surfactant complex formation depended upon the nature and concentration of the solubilized protein and on the surfactant concentration. Calculations of micellar occupancy and the relative surface areas of protein molecules and surfactant head-groups suggested that it was the exposure of the solubilized protein to the bulk organic solvent which promoted protein-surfactant complex formation as [P](rm) --> [P](rm,max). In the light of the experimental results and calculations described above, a mechanistic model is proposed to account for the observed phenomena. This is based upon the competing effects of increasing the solubilized protein concentration and the corresponding increase in the rate of protein-surfactant complex formation. The dynamic nature of the reverse micelles is inherent in the model, explaining the formation of the interfacial precipitate with time and its dependence on the internal phase volume of the micellar phase. Experiments on the co-partitioning of water and measurement ofthe AOT concentration in both phases verified the loss of protein, water, and surfactant from the organic phase at high values of [P](rm). (c) 1995 John Wiley & Sons Inc.     

Effect of pH on the transient reduction of pig-plasma benzylamine oxidase by benzylamine derivatives.

1. The transient kinetics of reduction of the 470-nm absorption band in benzylamine oxidase by substrate at different pH values between 6 and 10 have been studied by stopped-flow techniques, and substituent effects on kinetic parameters for the reduction process have been examined using a series of ring-substituted benzylamine derivatives as the substrates. 2. Reduction of the enzyme by substrate takes place in two kinetically distinguishable steps, with the intermediate formation of an enzyme-substrate complex in which the substrate appears to be covalently bound through its amino group to the prosthetic group of the enzyme, possibly in the form of an amine-pyridoxal Schiff-base. 3. The apparent stability of the enzyme-substrate complex shows no obvious dependence on the electronic properties of the amine substrates, but is strongly pH-dependent in a way suggesting that substrate-binding involves the non-protonated amines, exclusively, and requires the presence of the acid form of an ionizing group in the enzyme with apparent pKa of 8.8. 4. Reduction of the enzymatic 470-nm chromophore and release of the aldehyde product of the catalytic process are rate-limited by the same monomolecular reaction step involving the enzyme-substrate complex. Rate constants for the rate-limiting reaction exhibit no significant dependence on pH between 6 and 10, but correlate with Hammett sigma-values for the ring-substituted benzylamine derivatives tested, yielding a phi-value of + 0.3.     

Downstream protein separation by surfactant precipitation: a review

In a conventional protein downstream processing (DSP) scheme, chromatography is the single most expensive step. Despite being highly effective, it often has a low process throughput due to its semibatch nature, sometimes with nonreproducible results and relatively complex process development. Hence, more work is required to develop alternative purification methods that are more cost-effective, but exhibiting nearly comparable performance. In recent years, surfactant precipitation has been heralded as a promising new method for primary protein recovery that meets these criteria and is a simple and cost-effective method that purifies and concentrates. The method requires the direct addition of a surfactant to a complex solution (e.g. a fermentation broth) containing the protein of interest, where the final surfactant concentration is maintained below its critical micelle concentration (CMC) in order to allow for electrostatic and hydrophobic interactions between the surfactant and the target protein. An insoluble (hydrophobic) protein–surfactant complex is formed and backextraction of the target protein from the precipitate into a new aqueous phase is then carried out using either solvent extraction, or addition of a counter-ionic surfactant. Importantly, as highlighted by past researchers, the recovered proteins maintain their activity and structural integrity, as determined by circular dichroism (CD). In this review, various aspects of surfactant precipitation with respect to its general methodology and process mechanism, system parameters influencing performance, protein recovery, process selectivity and process advantages will be highlighted. Moreover, comparisons will be made to reverse micellar extraction, and the current drawbacks/challenges of surfactant precipitation will also be discussed. Finally, promising directions of future work with this separation technique will be highlighted.     

High pressure enzyme kinetics of dextransucrase.

The pressure dependence of enzymatic dextran formation has been observed up to 1000 at for several substrate concentrations. First order denaturation effects could be separated from the thermodynamic effects, which lead to a volume of 30.4 to 44.0 ccm per mole for the formation and -13.6ccm per mole for the activation of the enzyme-substrate complex. Denaturation depends on the substrate concentration. This leads to the conslusion that only the free enzyme is denatured, wheras the ES complex is stable.     

Molecular modeling of formate dehydrogenase: the formation of the Michaelis complex

The formation of the reactive enzyme-substrate complex of formate dehydrogenase has been investigated by molecular dynamics techniques accounting for different conformational states of the enzyme. Simulations revealed that the transport of substrate to the active site through the substrate channel proceeds in the open conformation of enzyme due to the crucial role of the Arg284 residue acting as a vehicle. However, formate binding in the active site of the open conformation leads to the formation of a nonproductive enzyme-substrate complex. The productive Michaelis complex is formed only in the closed enzyme conformation after the substrate and coenzyme have bound, when required rigidity of the binding site and reactive formate orientation due to interactions with Arg284, Asn146, Ile122, and His332 residues is attained. Then, the high occupancy (up to 75%) of the reactive substrate-coenzyme conformation is reached, which was demonstrated by hybrid quantum mechanics/molecular mechanics simulations using various semiempirical Hamiltonians.     

The role of the tryptophan-62 residue in the structure and function of lysozyme]

The thermostability and thermodinamics of formation of the enzyme-substrate complex of two oxidation products of chicken egg lysozyme with the tryptophane-62 residue modified to N'-formylkinurenine (with 2.5% activity) and kinurenine (with 27.5% activity) have been studied. In thermostability and pH effect on the substrate binding the lysozyme oxidation products do not differ from native lysozyme. The data obtained and thermodynamical characteristics of the enzyme-substrate complex formation suggest that the chemical nature of the 62 residue does not significantly affect the conformational properties of lysozyme, however, having a strongly pronounced effect on the binding of substrate and hence the total enzyme activity.     

Mechanism and binding specificity of beta-glucosidase-catalyzed hydrolysis of cellobiose analogues studied by competition enzyme kinetics monitored by 1H-NMR spectroscopy

The application of high-resolution 1H-NMR spectroscopy to monitor substrate and product time dependencies in progress curve enzyme kinetics is described with beta-glucosidase-catalyzed hydrolyses of cellobiose analogues as examples. It is demonstrated that inhibition patterns, relative binding specificities and catalytic rates can be inferred from competition experiments with two or more substrates. It could be concluded from competition experiments that substrates which form less stable enzyme-substrate complexes than methyl beta-cellobioside are hydrolyzed faster than this reference substrate when they are the sole substrate, due to a lower activation energy in the catalytic step, but that they are hydrolyzed slower than the reference compound in direct competition, due to the formation of the less stable enzyme-substrate complex in the binding step.     

Crystal structure of the reduced form of p-hydroxybenzoate hydroxylase refined at 2.3 A resolution.

The crystal structure of the reduced form of the enzyme p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens, complexed with its substrate p-hydroxybenzoate, has been obtained by protein X-ray crystallography. Crystals of the reduced form were prepared by soaking crystals of the oxidized enzyme-substrate complex in deaerated mother liquor containing 300-400 mM NADPH. A rapid bleaching of the crystals indicated the reduction of the enzyme-bound FAD by NADPH. This was confirmed by single crystal spectroscopy. X-ray data to 2.3 A were collected on oscillation films using a rotating anode generator as an X-ray source. After data processing and reduction, restrained least squares refinement using the 1.9 A structure of the oxidized enzyme-substrate complex as a starting model, yielded a crystallographic R-factor of 14.8% for 11,394 reflections. The final model of the reduced complex contains 3,098 protein atoms, the FAD molecule, the substrate p-hydroxybenzoate and 322 solvent molecules. The structures of the oxidized and reduced forms of the enzyme-substrate complex were found to be very similar. The root-mean-square discrepancy for all atoms between both structures was 0.38 A. The flavin ring is almost completely planar in the final model, although it was allowed to bend or twist during refinement. The observed angle between the benzene and the pyrimidine ring is 2 degrees. This value should be compared with observed values of 10 degrees for the oxidized enzyme-substrate complex and 19 degrees for the enzyme-product complex. The position of the substrate is virtually unaltered with respect to its position in the oxidized enzyme. No trace of a bound NADP+ or NADPH molecule was found.     

The pH dependence of kinetic isotope effects in monoamine oxidase A indicates stabilization of the neutral amine in the enzyme-substrate complex

A common feature of all the proposed mechanisms for monoamine oxidase is the initiation of catalysis with the deprotonated form of the amine substrate in the enzyme-substrate complex. However, recent steady-state kinetic studies on the pH dependence of monoamine oxidase led to the suggestion that it is the protonated form of the amine substrate that binds to the enzyme. To investigate this further, the pH dependence of monoamine oxidase A was characterized by both steady-state and stopped-flow techniques with protiated and deuterated substrates. For all substrates used, there is a macroscopic ionization in the enzyme-substrate complex attributed to a deprotonation event required for optimal catalysis with a pK(a) of 7.4-8.4. In stopped-flow assays, the pH dependence of the kinetic isotope effect decreases from approximately 13 to 8 with increasing pH, leading to assignment of this catalytically important deprotonation to that of the bound amine substrate. The acid limb of the bell-shaped pH profile for the rate of flavin reduction over the substrate binding constant (k(red)/K(s), reporting on ionizations in the free enzyme and/or free substrate) is due to deprotonation of the free substrate, and the alkaline limb is due to unfavourable deprotonation of an unknown group on the enzyme at high pH. The pK(a) of the free amine is above 9.3 for all substrates, and is greatly perturbed (DeltapK(a) approximately 2) on binding to the enzyme active site. This perturbation of the substrate amine pK(a) on binding to the enzyme has been observed with other amine oxidases, and likely identifies a common mechanism for increasing the effective concentration of the neutral form of the substrate in the enzyme-substrate complex, thus enabling efficient functioning of these enzymes at physiologically relevant pH.     

Enzyme kinetics: Inhibition equations derived from the inelastic collision hypothesis

The inelastic collision hypothesis of enzyme action has been proposed by G. Medwedew. This theory has been here extended to the cases of competitive and non-competitive inhibitors, which form enzyme-inhibitor complexes, and to the cases of competitive and non-competitive substrates. The resulting equations are discussed and contrasted to those derived from the classical enzyme-substrate hypothesis. The original formulation of Medwedew neglects the presence of the enzyme-substrate complex although the general theory admits the formation of this complex. The formulation has been revised and extended to include complex formation. The resulting equation is discussed in terms of the usual criteria used to evaluate the Michaelis-Menten-Briggs-Haldane equation. Taken in part from a thesis submitted by Robert Katzman to the faculty of the Department of Physiology, University of Chicago, in partial fulfillment of the requirements for the Degree of Master of Science.     

The determination of enzyme-substrate dissociation rates by dynamic isotope exchange enhancement experiments

A new method for the determination of dissociation rates of enzyme-substrate complexes has been developed. The rate of exchange of a labeled product back into the substrate is measured during catalysis of the forward reaction when the forward reaction is kept far from equilibrium by the enzymatic removal of the nonexchanging product. The ratio of the exchange rate and the net rate for product formation is then determined at various concentrations of the exchanging product. A plot of this ratio is a diagnostic indication of the kinetic mechanism and the relative rates of product dissociation from the binary and ternary enzyme complexes. This technique has been applied to the reaction catalyzed by bovine liver argininosuccinate lyase. The ratio for the rate of exchange of fumarate into argininosuccinate and the net rate for product formation was found to increase with the concentration of fumarate but to reach a limit of 3.3. The ratio of rates was half-maximal at 36 mM fumarate. The data have been interpreted to indicate the argininosuccinate lyase has a random kinetic mechanism. The calculated lower limit for the rate of release of arginine from the enzyme-fumarate-arginine complex is 0.35 times as fast as the Vmax in the reverse direction. The rate of release of arginine from the enzyme-arginine binary complex is 210 times faster than Vmax in the reverse direction.     

Regulation of alpha-ketoglutarate dehydrogenase cooperative properties in substrate binding by thiol-disulfide exchange

The influence of reducing the KGD non-cooperative form by DTT on the KG binding by the enzyme was investigated. The chemical modification of KGD by DEP has revealed that reduction of KGD cysteine residues results in the appearance of the interaction of the dimer active sites upon the enzyme-substrate complex formation. The reduction of 2 SH-groups per KGD subunit: the most reactive one and a buried one--was established to be sufficient for the appearance of KGD cooperative properties in substrate binding as well as for the change in the enzyme activity plots versus substrate concentration. It is suggested that KGD can be regulated by thiol-disulfide exchange in the cell.     

The energy landscape for ubihydroquinone oxidation at the Q(o) site of the bc(1) complex in Rhodobacter sphaeroides

Activation energies for partial reactions involved in oxidation of quinol by the bc(1) complex were independent of pH in the range 5. 5-8.9. Formation of enzyme-substrate complex required two substrates, ubihydroquinone binding from the lipid phase and the extrinsic domain of the iron-sulfur protein. The activation energy for ubihydroquinone oxidation was independent of the concentration of either substrate, showing that the activated step was in a reaction after formation of the enzyme-substrate complex. At all pH values, the partial reaction with the limiting rate and the highest activation energy was oxidation of bound ubihydroquinone. The pH dependence of the rate of ubihydroquinone oxidation reflected the pK on the oxidized iron-sulfur protein and requirement for the deprotonated form in formation of the enzyme-substrate complex. We discuss different mechanisms to explain the properties of the bifurcated reaction, and we preclude models in which the high activation barrier is in the second electron transfer or is caused by deprotonation of QH(2). Separation to products after the first electron transfer and movement of semiquinone formed in the Q(o) site would allow rapid electron transfer to heme b(L). This would also insulate the semiquinone from oxidation by the iron-sulfur protein, explaining the efficiency of bifurcation.     

Mixed micelles of monomeric and dimeric cationic surfactants with phospholipids: effect of hydrophobic interactions

Surface tension (gamma) and time resolved fluorescence quenching (TRFQ) measurements have been performed on the binary mixtures of monomeric as well as dimeric alkylammonium bromides with l-alpha-dimyristoylphosphatidycholine (DMPC) and L-alpha-dipalmitoylphosphatidycholine (DPPC). The critical micelle concentration (cmc) has been evaluated from the gamma measurements. The gamma plots show two breaks in the gamma versus [total surfactant] curves in most of the cases. The first break (C1) has been attributed to the mixed vesicle formation process. The break down of the vesicles leads to the mixed micellization between the surfactant and phospholipid monomers at the second break (C2). The amount of surfactant used in the vesicle breakdown process (DeltaC) increases linearly with the increase in the amount of phospholipid and depends significantly on the hydrophobicities of the cationic components. The surface area per molecule (a) evaluated from the gamma plots indicates compact monolayer formation in the case of monomeric surfactants with lower hydrophobicities and reverse is observed for dimeric surfactants. The pyrene life time (tau) of the solubilized pyrene in the hydrophobic environment of mixed micelles, fully supports the conclusion that derived from a.     

Use of reverse micelles in membrane protein structural biology

Membrane protein structural biology is a rapidly developing field with fundamental importance for elucidating key biological and biophysical processes including signal transduction, intercellular communication, and cellular transport. In addition to the intrinsic interest in this area of research, structural studies of membrane proteins have direct significance on the development of therapeutics that impact human health in diverse and important ways. In this article we demonstrate the potential of investigating the structure of membrane proteins using the reverse micelle forming surfactant dioctyl sulfosuccinate (AOT) in application to the prototypical model ion channel gramicidin A. Reverse micelles are surfactant based nanoparticles which have been employed to investigate fundamental physical properties of biomolecules. The results of this solution NMR based study indicate that the AOT reverse micelle system is capable of refolding and stabilizing relatively high concentrations of the native conformation of gramicidin A. Importantly, pulsed-field-gradient NMR diffusion and NOESY experiments reveal stable gramicidin A homodimer interactions that bridge reverse micelle particles. The spectroscopic benefit of reverse micelle-membrane protein solubilization is also explored, and significant enhancement over commonly used micelle based mimetic systems is demonstrated. These results establish the effectiveness of reverse micelle based studies of membrane proteins, and illustrate that membrane proteins solubilized by reverse micelles are compatible with high resolution solution NMR techniques. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Conformational aspects of beta-trypsin interaction with substrates and pancreatic trypsin inhibitor. I. Conformational properties of residues in the enzyme-active center and the structure of a non-valent enzyme-substrate complex

The theoretical conformational analysis of potential surfaces of Ser-195, His-57, Asp-102 and Gln-192 side chains in the active center of native beta-trypsin has been carried out. The above residues are shown to exist in low-energy conformational states in the free enzyme and in its nonbonded substrate complexes. Interrelations between the flexibility of the residues and their catalytical functions were revealed. Conformational aspects of interaction of trypsin with N-acetyl-L-lysine and N-acetyl-L-alanyl--L-alanyl--L-lysyl--L-alanyl methyl amide and with the Gly-12--Ile-19 BPTI fragment were analysed. The productive binding of the substrate at the nonbonded complex stage is shown to take place exclusively in the lowest energy conformation of the enzyme-substrate complex. Basing on theoretical and experimental evidence, the problems of primary and secondary specificity of trypsin, and potential properties of the native protein to form a productive nonbonded complex and to react at the subsequent stages of the catalytical act are discussed. Conformational changes in the active center and interactions with a substrate are shown to result from stabilizing enzyme-substrate interactions. Trypsin inhibition by BPTI molecule does not take place at the nonbonded complex stage.     

Purification of glycoproteins by selective transport using concanavalin-mediated reverse micellar extraction.

A novel methodology for coupling liquid-liquid extraction with affinity interaction has been developed to selectively and efficiently purify and separate glycoproteins. The basis for the separation is the selective extraction of glycoproteins from an aqueous solution into a reverse micellar organic phase by using concanavalin A (a sugar-binding lectin) as a facilitative carrier. Specifically, horseradish peroxidase (a common glycoprotein) can be bound to concanavalin A in an aqueous phase and then extracted into an AOT-isooctane organic phase with negligible loss in enzyme activity. Virtually no extraction of peroxidase occurs in the absence of concanavalin A. Electron spin resonance studies have shown that the large lectin-glycoprotein complex (96,000 daltons) resides in a nonaqueous environment within the reverse micelle, perhaps at the surfactant, water-pool interface; hence, extraction of the large complex is feasible. The facilitative extraction has been extended to selective transport of peroxidase from a mixture of peroxidase and alkaline phosphatase (a nonglycosylated protein). This results in an efficient separation strategy with a separation factor of 16.     

Kinetic specificity in papain-catalysed hydrolyses

The specificity of the proteolytic enzyme, papain, for the peptide bond of the substrate adjacent to that about to be cleaved and for the acyl residue of some N-acylglycine derivatives is manifest almost exclusively in the formation of the acyl-enzyme from the enzyme-substrate complex. Models for the enzyme-substrate complex and acyl-enzyme intermediate are suggested that account for these observations. In particular it is suggested that the peptide bond of the substrate adjacent to that about to be cleaved, is bound in the cleft of the enzyme between the NH group of glycine-66 and the backbone C=O group of aspartic acid-158, and provides a sensitive amplification mechanism through which the specificity of the enzyme for hydrophobic amino acids such as l-phenylalanine is relayed. It is also suggested that the distortion in the enzyme-substrate complex and the binding of the peptide bond adjacent to that about to be cleaved are also linked and behave co-operatively, the distortion of the protein facilitating binding and the stronger binding facilitating distortion. The results imply that between the enzyme-substrate complex and the acyl-enzyme a relaxation of the protein conformation must occur.