Effects of chemical modification on enzymatic activities and stabilities

The influence of chemical modification on the initial specific activity, residual activity, and deactivation kinetics of various enzymes is analyzed using a series mechanism. This straightforward multistate sequential model presented is consistent with the enzyme deactivation data obtained from different fields. The enzymes are placed in five different categories depending on the effect of chemical modification on initial specific activity and residual activity or stability. Wherever possible, structure-function relationships are described for the enzymes in the different categories. The categorization provides one avenue that leads to further physical insights into enzyme deactivation processes and into the enzyme structure itself.     

Categorization of enzyme deactivations using a series-type mechanism

A series-type enzyme deactivation model involving an active enzyme precursor and a final enzyme state with possible non-zero activity is proposed to categorize enzyme deactivation curves. The enzyme activity is a weighted function of the active enzyme states. The deactivation curves may be broadly classified into two major categories wherein the activity is either always less than or it may be more than the initial activity for some time period. Data taken from the literature may be classified into 14 cases. Complex enzyme deactivation curves exhibiting enzyme stabilization and a flex are some of the features that are classified.     

Enzyme deactivation

Enzyme deactivation kinetics is often first-order. Different examples of first-order deactivation kinetics exhibited by different enzymes under a wide variety of conditions are presented. Examples of both soluble and immobilized enzymes are presented. The influence of different parameters, chemical modification of specific residues, inhibitors, inactivators, protecting agents, induced conformational changes by external agents, enzyme concentration, and different substrates on the first-order inactivation kinetics of different enzymes is analyzed. The different examples presented from a variety of different areas provides a judicious framework and collection demonstrating the wide applicability of first-order deactivation kinetics. Examples of reversible first-order deactivation kinetics and deactivation-disguise kinetics are also presented.Different mechanisms are also presented to model complex enzyme deactivations. The non-series type mechanisms are emphasized and these involve the substrate and chemical modifiers. Substrate-dependent deactivation rate expressions that are of "separable" and "non-separable" type are presented. Rate expressions involving time-dependent rate constants along with their corresponding mechanisms are presented. Examples of enzymes that exhibit a deactivation-free grace period are also given. An interesting case of enzyme inactivation is the loss of activity in the presence of an auto-decaying reagent. The method is presented by which the intrinsic inactivation rate constants may be obtained. Examples of pH-dependent enzyme inactivation are presented that may be modelled by a five-step (or a simplified two-step) mechanism, and also by a single-step mechanism involving residual activity for the final state. Appropriate examples of enzyme inactivation are presented in each case to highlight the different mechanisms involved.     

The effect of hydrophobization of glucose-6-phosphate dehydrogenase with progesterone on its thermal inactivation

Thermal inactivation of glucose-6-phosphate dehydrogenase (G6PDH) and its conjugates with progesterone containing 3, 7 and 35 molecules of the modifier was studied in bidistilled water over a temperature range 35-47 degrees. At different temperatures and initial concentrations of the enzyme and its modified forms, thermal inactivation is described by the equation of the first order up to a significant degree of enzyme deactivation. The effective Kin values are decreased with the increase of the native G6PDH concentration and changed in a complicated manner with the increase of the conjugate concentration depending on the enzyme modification degree, which reflects a great role of the enzyme hydrophobicity in its inactivation. The role of hydrophobicity of the modified G6PDH in changes of its specific activity is discussed.     

A mathematical analysis of enzyme stabilization by a series-type mechanism: Influence of chemical modifiers

A series-type enzyme deactivation model is utilized to theoretically analyze and to quantify the effect of chemical modifier concentration on the eventual level of enzyme activity stabilization, alpha(2). An increase in the concentration of phosphate ion and NADP increases alpha(2) for the enzymes studied. One example of each enzyme deactivation is given wherein the introduction of chemical modifiers changes the deactivation mechanism from a single-step to a series-type mechanism, and from a series-type to a single-step mechanism. Simple empirical equations are proposed to quantify the effect of chemical modifier concentration on alpha(2).     

An experimental method to determine the substrate protection of enzyme against deactivation in a reversible reaction.

The substrate protection effect on an enzyme in a reversible reaction was studied by using glucose isomerase immobilized in small particles (radius less than 100 micron). Deactivation of the enzyme at various substrate concentrations in Tris buffer, pH 8.25, at 62.1 degrees C was studied in eight-column reactor sets. At set times the immobilized enzyme in one of the eight reactors was taken out and rinsed thoroughly, and then its residual activity was determined. The conclusions are, first, that the protection by the reactant is equal to the protection by the product, and, secondly, that the half-life of the enzyme increases slowly at high sugar concentrations. Thus the experimental method described here appears to be a useful one for the determination of substrate protection of enzyme deactivation in reversible reactions.     

The chemical modification of alpha-chymotrypsin with both hydrophobic and hydrophilic compounds stabilizes the enzyme against denaturation in water-organic media.

We considered alpha-chymotrypsin (CT) in homogeneous water-organic media as a model system to examine the influence of enzyme chemical modification with hydrophilic and hydrophobic substances on its stability, activity and structure. Both types of modifying agents may lead to considerable stabilization of the enzyme in water-ethanol and water-DMF mixtures: (i) the range of organic cosolvent concentration at which enzyme activity (Vm) is at least 100% of its initial value is broadened and (ii) the range of organic cosolvent concentration at which the residual enzyme activity is observed is increased. We found that for both types of modification the stabilization effect can be correlated with the changes in protein surface hydrophobicity/hydrophilicity brought about by the modification. Circular dichroism studies indicated that the effects of these two types of modification on CT structure and its behavior in water-ethanol mixtures are different. Differential scanning calorimetry studies revealed that after modification two or three fractions or domains, differing in their stability, can be resolved. The least stable fractions (or domains) have properties similar to native CT.     

Influence of modifying agents on enzyme inactivation studies. An analysis using a series mechanism and a form of the Hill-type equations

A series deactivation model is utilized to theoretically examine the influence of different modifying agents on enzyme deactivation kinetics. A form of the Hill-type equation is used to describe the effect of the modifying agents on the model parameters. Modification-induced inactivation equations are presented for the acetylation and succinylation of E. Coli asparaginase, for the site-specific reagent and substrate modification of flavocytochrome b(2) from Baker's yeast, and for the guanidinium chloride inactivation of cathepsin D. The analysis of more data for these and other enzymes would help further substantiate the technique presented and enhance the applicability of the model.     

The possibility for improvement of ceramic membrane ultrafiltration of an enzyme solution

Ultrafiltration represents an attractive downstream processing technique for enzymes concentration and their primary purification. However, the process efficiency is often limited by protein fouling and shear-induced enzyme deactivation, resulting in permeate flux decline and loss of enzyme activity. The objective of this work was to investigate the possibility for improvement of ceramic membrane ultrafiltration of endo-pectinase solution. Experimental investigations were performed on a 5 nm ceramic membrane with the Kenics static mixer placed inside the membrane in order to improve the process performance. The use of the static mixer resulted in the flux improvement of about 45% at a volume concentration factor (VCF) of 3 leading to the reduction of operation time of 25% and the energy saving of about 40%. Although the rejection of endo-pectinase was higher than 96%, the extensive loss of the enzyme activity during operation indicated that the modification of the feed solution is essential for improved ultrafiltration performance. Addition of pectin to the original endo-pectinase solution led to a significant reduction of the enzyme deactivation: the enzyme activity yield was 90% at a VCF of 1.6 during operation with the static mixer.     

Stability index for enzymes deactivating by different mechanisms.

A quantitative procedure for estimating changes in enzyme stability upon chemical modification is presented. Stability index for different deactivation mechanisms is presented and applied to different enzyme deactivations. The stability index provides a convenient method of estimating changes in enzyme stability upon chemical modification.     

Equilibrium studies on the refolding and reactivation of rabbit-muscle aldolase after acid dissociation.

Dissociation, denaturation, and deactivation of aldolase from rabbit muscle in the acid pH range have been investigated using sedimentation analysis, fluorescence, circular dichroism, and activity tests. Under comparable experimental conditions the pH-dependent profiles of deactivation and denaturation parallel the dissociation of the enzyme. In the range of dissociation at pH4-5tetramers and monomers are in equilibrium. Intrinsic chromophores and far-ultraviolet circular dichroism suggest the transition to be a complex multistep process. At pH approximately 2.3 the enzyme is split into its fully inactive monomers which still contain some residual secondary structure. After reassociation under optimum conditions (0.2 M phosphate buffer pH 7.6, 1 mM EDTA, 0.1 mM dithiothreitol, 0 degrees C, enzyme concentration 0.4-59 mug/ml) up to 95% enzymic activity is recovered which belongs to a renatured tetrameric species indistinguishable from the native enzyme by all available biochemical and physicochemical criteria.     

Deactivation of isoamylase and β-amylase in the agitated reactor under supercritical carbon dioxide

The current research examines the impact of agitation on deactivation of isoamylase and β-amylase under supercritical carbon dioxide (SC-CO₂). Our experimental results showed that the activity of either enzyme decreased with increasing pressure or speed of agitation. The degree of enzymatic deactivation caused by pressure became more prominent in the presence of agitation, suggesting that the agitation plays an important role in enzymatic deactivation in SC-CO₂ environment. Moreover, the enzymatic deactivation behavior associated with agitation and pressure was further quantitatively analyzed using a proposed inactivation kinetic model. Our analysis indicated that isoamylase and β-amylase exhibited significantly different relationships between the inverse of percentage residual activity and the product of number of revolution per time and time elapsed under pressurized carbon dioxide. We believe that the outcome from this work may provide a better understanding of the effects of agitation and pressure in enzyme deactivation behavior under SC-CO₂.     

Functional arginine residues involved in coenzyme binding by glutamate dehydrogenases.

Reaction of the NADP-dependent glutamate dehydrogenase of Neurospora with 1,2-cyclohexanedione results in a biphasic loss of enzyme activity. At the end of the rapid phase of the reaction (t1/2 = 1.5 min) the enzyme activity is diminished by approximately 60% with the simultaneous loss of 1 residue of arginine per subunit. After 60 min, the enzyme activity is completely lost with the modification of a total of 2 arginine residues per subunit. Reaction of bovine liver glutamate dehydrogenase with cyclohexanedione causes a rapid loss of approximately 45% of the enzyme activity and modification of about 1.5 residues of arginine per subunit. More prolonged treatment results in reaction of an additional 4 residues of arginine per subunit but is without further effect on the residual activity. The activity of the Neurospora enzyme is not protected by substrate, coenzyme, or a combination of both; however, the activity of the bovine enzyme is partially protected by high levels of NAD or NADP. Although the Km for alpha-ketoglutarate is unchanged by a limited modification of either enzyme with cyclohexanedione, the Km for coenzyme is increased about 2-fold for the Neurospora enzyme and about 1.5-fold for the bovine enzyme. The Ki of the Neurospora dehydrogenase for the competitive inhibitor 2'-monophosphoadenosine-5'-diphosphoribose is unchanged by the enzyme modification, but nicotinamide mononucleotide, a competitive inhibitor for the native Neurospora enzyme, does not inhibit the glutamate dehydrogenase with 1 modified arginine residue. This finding implies that the modified arginine is at or near the nicotinamide binding iste of the enzyme.     

Series-type enzyme deactivations: Influence of intermediate activity on deactivation kinetics

A series-type enzyme deactivation model involving an active enzyme precursor is proposed wherein the enzyme activity is a weighted function of the active enzyme states. The active enzyme precursor may be less active, as active or more active than the initial enzyme form. The proposed model is shown to fit the soluble and immobilized enzyme deactivation data presented reasonably well. Some enzymes exhibit a ‘compensation-like’ effect. In other enzymes, if the deactivation rate coefficient for the second step, k₂, is zero, then the activity may stabilize to a value that depends upon the relative activities of the two active enzyme states.     

The calmodulin-dependent activation and deactivation of the phosphoprotein phosphatase, calcineurin, and the effect of nucleotides, pyrophosphate, and divalent metal ions. Identification of calcineurin as a Zn and Fe metalloenzyme

Studies on the interaction of calcineurin with its activator, calmodulin, showed that the 1:1 complex is the activated species. Concomitant with activation, a time-dependent deactivation of the phosphatase was observed. The process followed first order kinetics and was dependent on the presence of both Ca2+ and calmodulin. The deactivation rate constant at pH 7.6 and 30 degrees C was 0.06 min-1, which was increased by the substrate, p-nitrophenylphosphate (Km = 11 mM), to 0.47 min-1. PPi and nucleotides inhibited the enzyme competitively and accelerated the deactivation. The first order rate constant was increased to 2.3 min-1 by PPi (Ki = 55 microM) and to 8.0 min-1 by ADP (Ki = 0.94 mM). A theory dealing with the deactivation (applicable to chemical modification, etc.) of an enzyme in the absence and presence of various ligands is presented. The deactivated enzyme remained bound to calmodulin and was not reactivated by dissociation-reassociation of the calcineurin-calmodulin complex. Calcineurin was found to contain covalently bound phosphate; however, no difference in its content was detected upon deactivation, indicating that self-dephosphorylation was not involved. The deactivation could be reversed, as well as prevented, by divalent metal ions such as Ni2+ and Mn2+. Atomic absorption spectroscopy revealed nearly stoichiometric amounts of tightly bound Fe and Zn (but little other ions) on purified calcineurin, which remained bound during the calmodulin-dependent deactivation; removal of tightly bound metals is, therefore, not the cause of deactivation. Our results indicate that calcineurin is a metallophosphatase and not simply a Ca2+- and calmodulin-stimulated enzyme. In addition to the nondissociable Zn and Fe and the Ca2+ bound to the B subunit and calmodulin, the enzyme requires a divalent metal ion for structural stability and full activity.     

The modification of sulfhydryl groups of glutamine synthetase from Bacillus stearothermophilus with 5, 5'-dithiobis(2-nitrobenzoic acid).

The SH groups of glutamine synthetase [EC 6.3.1.2] from Bacillus stearothermophilus were modified with 5, 5'-dithiobis(2-nitrobenzoic acid) in order to determine the number of SH groups in the molecule as well as the effect of the modification on the enzyme activity. Three SH groups per subunit were detected after complete denaturation of the enzyme with 6 M urea, one of which was essential for the enzyme activity in view of its reactivity with 5, 5'-dithiobis(2-nitrobenzoic acid) on addition of MgCl2 with loss of the activity. The CD spectra of the modified enzyme in the near ultraviolet region changed from that of the native enzyme, indicating that aromatic amino acid residues were affected by modification of the SH group. The fluorescence derived from tryptophanyl residue(s) was quenched depending on the extent of modification of the SH group, suggesting that the tryptophanyl residue(s) was located in the proximity of the SH group. The thermostability of the enzyme was remarkably decreased by modification of the SH group.     

Effect of immobilized enzyme deactivation in fixed- and fluid-bed reactors

A two-parameter theoretical model is developed to evaluate the effect of immobilized enzyme deactivation on substrate conversion in fixed- and fluid-bed reactors under diffusion-free conditions. The method describes a simple reaction in which three different immobilized enzyme deactivation forms are considered, and an expression is developed to evaluate the effect of immobilized enzyme deactivation on yield in a consecutive reaction. Comparison of reactor performances for the two reactor types reduces to a comparison of the appropriate dimensionless parameters. The practical implications of the development are illustrated through an example.     

Role of a disulfide bond in the gamma subunit in activation of the ATPase of chloroplast coupling factor 1

The relationship between activation of the latent ATPase activity of isolated chloroplast coupling factor 1 (CF1) and reduction of a disulfide in the gamma subunit has been assessed. The sulfhydryl residues involved in the disulfide bond are distinct from residues normally accessible to maleimide modification during incubation of thylakoids in the dark or the light. Dithiothreitol-induced activation is time dependent, and correlates with reduction of the disulfide. Sulfhydryl residues exposed during activation can be reoxidized to disulfide by incubation with iodosobenzoate , with a concomitant loss of ATPase activity. Activation and deactivation are reversible, but deactivation is prevented by treatment of the reduced enzyme with N-ethylmaleimide. Heat activation does not reduce the disulfide bond unless dithiothreitol is present during activation. Prior heating of CF1, which partially activates the enzyme, renders the disulfide more susceptible to subsequent dithiol reduction. The activity obtained when heat and dithiothreitol are used together is approximately equal to the sum of the partial activations obtained with heat or dithiothreitol alone. Iodosobenzoate has no effect on heat-activated CF1. Enzyme activated by heating in the presence of dithiothreitol can be partially deactivated, consistent with reversal of the activity attributable to the dithiol effect. Fluorescence polarization of anilinonaphthylmaleimide bound to the reduced enzyme indicates that the sulfhydryl residues involved in the disulfide are in a less rigid environment than the other two sulfhydryl residues in the gamma subunit. Polarization of anilinonaphthylmaleimide bound to these sulfhydryls is reduced by heat treatment of CF1. The increased susceptibility of the disulfide to reduction upon heat treatment, and the activation of ATPase activity with or without disulfide bond cleavage are indicative of conformational changes within the gamma subunit that occur during the conversion of CF1 from a latent to an active ATPase. In addition the results are consistent with at least two distinct conformational forms of CF1 that can hydrolyze ATP.     

Prediction of continuous reactors performance based on batch reactor deactivation kinetics data of immobilized lipase

Experiments on deactivation kinetics of immobilized lipase enzyme fromCandida cylindracea were performed in stirred batch reactor using rice bran oil as the substrate and temperature as the deactivation parameter. The data were fitted in first order deactivation model. The effect of temperature on deactivation rate was represented by Arrhenius equation. Theoretical equations were developed based on pseudo-steady state approximation and Michaelis-Menten rate expression to predict the time course of conversion due to enzyme deactivation and apparent half-life of the immobilized enzyme activity in PFR and CSTR under constant feed rate policy for no diffusion limitation and diffusion limitation of first order. Stability of enzyme in these continuous reactors was predicted and factors affecting the stability were analyzed.     

Microheterogeneity of enzymes and deactivation

The influence of microheterogeneity on enzyme inactivation kinetics is presented. Examples of different enzymes are given where microheterogeneity has been detected by different techniques. The different statistical models are presented which include the influence of microheterogeneity on enzyme inactivation kinetics and stability. As the microheterogeneity of the enzyme increases, there is a sharper decline in the normalized activity during the initial stages of the deactivation but a greater stability and activity, compared to similar homogeneous enzyme, as the deactivation proceeds. Microheterogeneity makes the deactivation reaction have a higher apparent order of reaction. The implications of microheterogeneity on enzyme inactivations are high lighted by different examples. The analysis provides fresh physical insights into the chemistry, subpopulations, structure, and function of enzymes.