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.     

Kinetic analysis of the general modifier mechanism of Botts and Morales involving a suicide substrate

Suicide substrates are widely used in enzymology for studying enzyme mechanisms and designing potential drugs. The presence of a reversible modifier decreases or increases the rate of substrate-induced inactivation, with evident physiological and experimental consequences. To date, only the action of a competitive or uncompetitive inhibitor of an enzyme system involving suicide substrate has been reported. In this paper, we analyse the kinetics of enzyme-catalysed reactions which evolve in accordance with the general modifier mechanisms of Botts and Morales in which enzyme inactivation is induced by suicide substrate. Rapid equilibrium of all of the reversible reaction steps involved is assumed and the time course equations for the residual enzyme activity, the inactive enzyme forms and the reaction product are derived. Partition ratios giving the relative weight of the product and inactive enzyme concentrations, and the relative contribution to the product formation of each of the unmodified and modified catalytic routes, are studied. New indices pointing to the conditions under which the modifier acts as inhibitor or as activator are suggested. The goodness of the analytical solutions is tested by comparison with the simulated curves obtained by numerical integration. An experimental design and kinetic data analysis to evaluate the kinetic parameters from the time progress curves of the product are proposed. From these results, those corresponding to several reaction mechanisms involving both a suicide substrate and a modifier, and which can be regarded as particular cases of the general case analysed here, can be directly and easily derived.     

Some kinetic studies on the mechanism of action of carnitine acetyltransferase

1. Michaelis constants for substrates of carnitine acetyltransferase have been shown to be independent of the concentration of second substrate present. This applies to the forward reaction between acetyl-l-carnitine and CoASH, and to the back reaction between l-carnitine and acetyl-CoA. 2. Product inhibition of both forward and back reactions has been studied. Evidence has been obtained for independent binding sites for l-carnitine and CoASH. Acetyl groups attached to either substrate occupy overlapping positions in space when the substrates are bound to the enzyme. 3. Possible reaction mechanisms involving the ordered addition of substrates have been excluded by determining kinetic constants in the presence and absence of added product. 4. d-Carnitine and acetyl-d-carnitine have been shown to inhibit competitively with respect to l-carnitine and acetyl-l-carnitine. 5. It is concluded that the mechanism of action of carnitine acetyltransferase involves four binary and two or more ternary enzyme complexes in rapid equilibrium with free substrates, the interconversion of the ternary complexes being the rate-limiting step. The possible intermediate formation of an acetyl-enzyme cannot be excluded, but this could only arise from a ternary complex.     

Constant-ratio alternative substrate approach for differentiation of enzyme mechanisms

A new kinetic approach using alternative substrates as a tool for studying enzyme mechanisms is described. In this method the substrate to alternative substrate ratio is maintained constant and the common product (or summation of product analogs) is measured. The double-reciprocal plots so obtained at several constant ratios generate different patterns for various mechanisms, thus permitting a choice of kinetic model. In some cases, secondary intercept plots are utilized as a diagnostic aid. Another feature of this approach is that most of the resultant plots are linear. The graphical patterns for four cases of two-substrate, two-product reactions are presented as examples. These patterns allow one to differentiate several mechanisms which are not distinguishable by conventional alternative substrate, competitive inhibitor, or product inhibition studies alone. When used in combination with other methods, various mechanisms involving isomerization and abortive complex formation can be differentiated even if only one alternative substrate is available.     

Initiation and inhibition of free-radical processes in biochemical peroxidase systems: a review

The role of complexes containing oxygen or peroxide in monooxygenase systems and models thereof, as well as in peroxidase- and quasi-peroxidase-catalyzed processes, has been reviewed. Pathways of conversion of these intermediate complexes involving single-electron (radical) and two-electron (heterolytic) mechanisms are dealt with. Coupled peroxidase-catalyzed oxidation of aromatic amines and phenols is analyzed; inhibition and activation of peroxidase-catalyzed reactions are characterized quantitatively. Oxidation of chromogenic substrates (ABTS, OPD, and TMB) in the presence of phenolic inhibitors or polydisulfides of substituted phenols is characterized by inhibition constants (Ki, micromol). Activation of peroxidase-catalyzed oxidation of the same substrates is characterized by the degree (coefficient) of activation (alpha, M(-1)), which was determined for 2-aminothiazole, melamine, tetrazole, and its 5-substituted derivatives. Examples of applied use of peroxidase-catalyzed enzyme and model systems are given (oxidation of organic compounds, chemical analysis, enzyme immunoassay, tests for antioxidant activity of biological fluids).     

Calculation of steady-state rate equations and the fluxes between substrates and products in enzyme reactions.

1. Two methods are described for deriving the steady-state velocity of an enzyme reaction from a consideration of fluxes between enzyme intermediates. The equivalent-reaction technique, in which enzyme intermediates are systematically eliminated and replaced by equivalent reactions, appears the most generally useful. The methods are applicable to all enzyme mechanisms, including three-substrate and random Bi Bi Ping Pong mechanisms. Solutions are obtained in algebraic form and these are presented for the common random Bi Bi mechanisms. The steady-state quantities of the enzyme intermediates may also be calculated. Additional steps may be introduced into enzyme mechanisms for which the steady-state velocity equation is already known. 2. The calculation of fluxes between substrates and products in three-substrate and random Bi Bi Ping Pong mechanisms is described. 3. It is concluded that the new methods may offer advantages in ease of calculation and in the analysis of the effects of individual steps on the overall reaction. The methods are used to show that an ordered addition of two substrates to an enzyme which is activated by another ligand will not necessarily give hyperbolic steady-state-velocity kinetics or the flux ratios characteristic of an ordered addition, if the dissociation of the ligand from the enzyme is random.     

How does parkin ligate ubiquitin to Parkinson's disease?

Recessive mutations in the human PARKIN gene are the most common cause of hereditary parkinsonism, which arises from the degeneration of dopaminergic neurons in the substantia nigra. However, the molecular mechanisms by which the loss of parkin causes dopaminergic neurodegeneration are not well understood. Parkin is an enzyme that ubiquitinates several candidate substrate proteins and thereby targets them for proteasomal degradation. Hypothesis-driven searches have led to the discovery of aggregation-prone protein substrates of parkin. Moreover, the enzyme is upregulated when under unfolded protein stress. Thus, loss-of-function mutations of parkin might impair the removal of potentially toxic protein aggregates. However, the limited neuropathological information that is available from parkin-proven patients, as well as the recent knockout of the parkin gene in fruit flies and mice, may indicate a more complex disease mechanism, possibly involving the misfolding of parkin itself or of additional substrates. The risk factors that predispose dopaminergic neurons to degenerate on parkin failure are yet to be identified.     

The role of NAD(P)H oxidoreductase (DT-Diaphorase) in the bioactivation of quinone-containing antitumor agents: a review

Bioactivation of quinone-containing anticancer agents has been studied extensively within the context of the chemistry and structure of the individual quinones which may result in various mechanisms of bioactivation and activity. In this review we focus on the two electron enzymatic reduction/activation of quinone-containing anticancer agents by DT Diaphorase (DTD). This enzyme has become important in oncopharmacology because its activity varies with tissues and it has been found to be elevated in tumors. Thus, a selective tumor cell kill can exist for agents that are good substrates for this enzyme. In addition, the enzyme can be induced by a variety of agents, a fact that can be used in chemotherapy. That is induction by a nontoxic agent followed by treatment with a good DT-Diaphorase substrate. A wide variety of anticancer drugs are discussed some of which are not good substrates such as Adriamycin, and some of which are excellent substrates. The latter category includes a variety of quinone containing alkylating agents.     

Investigations of reaction kinetics for immobilized enzymes--identification of parameters in the presence of diffusion limitation

A method is proposed for identification of kinetic parameters when diffusion of substrates is limiting in reactions catalyzed by immobilized enzymes. This method overcomes conventional sequential procedures, which assume immobilization does not affect the conformation of the enzyme and, thus, consider intrinsic and inherent kinetics to be the same. The coupled equations describing intraparticle mass transport are solved simultaneously using numerical methods and are used for direct estimation of kinetic parameters by fitting modeling results to time-course measurements in a stirred tank reactor. While most traditional procedures were based on Michaelis-Menten kinetics, the method presented here is applicable to more complex kinetic mechanisms involving multiple state variables, such as ping-pong bi-bi. The method is applied to the kinetic resolution of (R/S)-1-methoxy-2-propanol with vinyl acetate catalyzed by Candida antarctica lipase B. A mathematical model is developed consisting of irreversible ping-pong bi-bi kinetics, including competitive inhibition of both enantiomers. The kinetic model, which fits to experimental data over a wide range of both substrates (5-95%) and temperatures (5-56 degrees C), is used for simulations to study typical behavior of immobilized enzyme systems.     

泛素链的形成机理

泛素化是真核细胞中重要的蛋白质翻译后修饰过程,通过靶向蛋白质降解或其他信号途径参与多种细胞功能.底物蛋白的多聚泛素化修饰是一个持续的过程,其中不仅涉及复杂泛素系统相关酶的参与,而且存在更为复杂的结构上相互作用与泛素链组装机理.不同的泛素链修饰决定了底物蛋白下游的不同命运,泛素结合酶E2在泛素链形成中的重要作用受到越来越多的关注.对泛素链形成机理的深入研究与认识有利于发现与泛素系统相关的疾病靶点和利用泛素化调控方法进行治疗.本综述总结了E2和E3如何决定不同泛素链形成的机制和相关的结构信息,以及两种不同的泛素链组装机制.     

Initiation and inhibition of free-radical processes in biochemical peroxide systems: A review

The role of complexes containing oxygen or peroxide in monooxygenase systems and models thereof, as well as in peroxidase-and quasi-peroxidase-catalyzed processes, has been reviewed. Pathways of conversion of these intermediate complexes involving single-electron (radical) and two-electron (heterolytic) mechanisms are dealt with. Peroxidase-catalyzed co-oxidation of aromatic amines and phenols is analyzed; inhibition and activation of peroxidase-catalyzed reactions are characterized quantitatively. Oxidation of chromogenic substrates (ABTS, OPD, and TMB) in the presence of phenolic inhibitors or polydisulfides of substituted phenols is characterized by inhibition constants (K _i, μmol). Activation of peroxidase-catalyzed oxidation of the same substrates is characterized by the degree (coefficient) of activation (α, M^?1), which was determined for 2-aminothiazole, melamine, tetrazole, and its 5-substituted derivatives. Examples of applied use of peroxidase-catalyzed enzyme and model systems are given (oxidation of organic compounds, chemical analysis, enzyme immunoassay, tests for antioxidant activity of biological fluids).     

"Apoptotic" mechanisms in normal brain plasticity: caspase-3 and long-term potentiation

The analysis of recent data indicates that a few enzymes that have been recognized as "apoptotic" so far may be involved in important cellular processes not related to cell death in the brain. For example, it can be demonstrated that caspase-3, an "apoptotic" enzyme that is active in neurons is necessary for normal neuroplasticity. Here we discuss the involvement of caspase-3 in long-term potentiation phenomenon. Proteins that are key players of molecular mechanisms of long-term potentiation induction and maintenance are also caspase-3 substrates. A concept on a new mechanism of synaptic plasticity modulation involving caspase-3 has been formulated postulating a specific role of caspase-3 in normal brain functioning.     

Catalytic mechanisms of glutamine synthetase enzymes. Studies with analogs of possible intermediates and transition states.

Glutamine synthetase enzymes isolated from pea seeds and from Escherichia coli are observed to behave differently in experiments designed to probe reaction mechanism. Although both enzymes were found to bind and release substrates in random order mechanisms (Wedler, F.C. (1974) J. Biol. Chem, 247, 5080-5087), isotopic exchanges with partial reaction systems indicative of a gamma-glutamylphosphate intermediate are catalyzed only by the pea seed enzyme. The E. coli system fails to catalyze any exchanges at appreciable rates unless all substrates are present. This negative result implies either an absolute conformational requirement for bound substrates or that the putative complex (E-Glu-P-MgADP) is exceedingly tight. To test the latter, a nonreactive structural analog of gamma-glutamyl-phosphate, namely 3-(phosphonoacetylamido)-L-alanine (PA2LA), has been synthesized. With the E. coli enzyme PA2LA was found to bind no more tightly than L-glutamate and is strictly competitive versus L-glutamate (Ki = 3 mM). Thus, failure to catalyze partial exchange reactions indicative of gamma-Glu-P is probably not attributable to tight complex formation. The binding of PA2LA with the pea seed enzyme apparently involves a two-step process: a rapid, reversible step in which PA2LA binds 10-fold more tightly than L-glutamate, followed by a slow (but reversible) process involving very tight PA2LA binding, apparently with enzyme isomerization promoted by nucleotide. The specificity of the two enzymes toward L-methionine-SR-sulfoximine, Met(O)(NH), was also different...     

Use of conformationally restricted pyridinium alpha-D-N-acetylneuraminides to probe specificity in bacterial and viral sialidases.

Investigations into subtle changes in the catalytic activity of sialidases have been performed using enzymes from several different origins, and their results have been compared. This work highlights the potential pitfalls encountered when extending conclusions derived from mechanistic studies on a single enzyme even to those with high-sequence homology. Specifically, a panel of 5 pyridinium N-acetylneuraminides were used as substrates in a study that revealed subtle differences in the catalytic mechanisms used by 4 different sialidase enzymes. The lowest reactivity towards the artificial (pyridinium) substrates was displayed by the Newcastle disease virus hemagglutinin-neuraminidase. Moreover, in reactions involving aryl N-acetylneuraminides, the activity of the Newcastle enzyme was competitively inhibited by the 3,4-dihydro-2H-pyrano[3,2-c]pyridinium compound with a Ki = 58 micromol/L. Alternatively, the 3 bacterial enzymes tested, from Salmonella typhimurium, Clostridium perfringens, and Vibrio cholerae, were catalytically active against all members of the panel of substrates. Based on the observed effect of leaving-group ability, it is proposed that the rate-determining step for kcat (and likely for kcat/Km as well) with each bacterial enzyme is as follows: sialylation, which is concerted with conformational change for V. cholerae; and conformational change for S. typhimurium and C. perfringens.     

Fixation of a highly reactive form of alpha-chymotrypsin by micellar matrix

Using reversed micelles of surfactants solvated by water-organic cosolvent mixtures as a matrix for enzyme entrapping, it is possible to fix the highly reactive alpha-chymotrypsin form. The reactivity of alpha-chymotrypsin towards nonspecific substrates increases to the extent comparable with that observed in reactions involving specific substrates.     

Probing the Activity Modification Space of the Cysteine Peptidase Cathepsin K with Novel Allosteric Modifiers

Targeting allosteric sites is gaining increasing recognition as a strategy for modulating the activity of enzymes, especially in drug design. Here we investigate the mechanisms of allosteric regulation of cathepsin K as a representative of cysteine cathepsins and a promising drug target for the treatment of osteoporosis. Eight novel modifiers are identified by computational targeting of predicted allosteric sites on the surface of the enzyme. All act via hyperbolic kinetic mechanisms in presence of low molecular mass substrates, as expected for allosteric effectors. Two compounds have sizable effects on enzyme activity using interstitial collagen as a natural substrate of cathepsin K and four compounds show a significantly stabilizing effect on cathepsin K. The concept of activity modification space is introduced to obtain a global perspective of the effects elicited by the modifiers. Analysis of the activity modification space reveals that the activity of cathepsin K is regulated via multiple, different allosteric mechanisms.     

Kinetics of three substrate enzyme systems. Treatment of partially random mechanisms using equilibrium assumptions

There are three partially random kinetic mechanisms which may be visualized for enzymes that catalyze reactions involving three substrates. The steady-state rate equation for these mechanisms can be reduced to the form derived by assuming equilibrium kinetics from consideration of inequality relationships among rate constants alone. The validity of these assumptions and of the resulting rate laws is considered.     

The effects of substrate composition, quantity, and diversity on microbial activity

Variation in organic matter inputs caused by differences in plant community composition has been shown to affect microbial activity, although the mechanisms controlling these effects are not entirely understood. In this study we determine the effects of variation in substrate composition, quantity, and diversity on soil extracellular enzyme activity and respiration in laboratory microcosms. Microbial respiration responded predictably to substrate composition and quantity and was maximized by the addition of labile substrates and greater substrate quantity. However, there was no effect of substrate diversity on respiration. Substrate composition significantly affected enzyme activity. Phosphatase activity was maximized with addition of C and N together, supporting the common notion that addition of limiting resources increases investment in enzymes to acquire other limiting nutrients. Chitinase activity was maximized with the addition of chitin, suggesting that some enzymes may be stimulated by the addition of the substrate they degrade. In contrast, activities of glucosidase and peptidase were maximized by the addition of the products of these enzymes, glucose and alanine, respectively, for reasons that are unclear. Substrate diversity and quantity also stimulated enzyme activity for three and four of the six enzymes assayed, respectively. We found evidence of complementary (i.e., non-additive) effects of additions of different substrates on activity for three of the six enzymes assayed; for the remaining enzymes, effects of adding a greater diversity of substrates appeared to arise from the substrate-specific effects of those substrates included in the high-diversity treatment. Finally, in a comparison of measures of microbial respiration and enzyme activity, we found that labile C and nutrient-acquiring enzymes, not those involved in the degradation of recalcitrant compounds, were the best predictors of respiration rates. These results suggest that while composition, quantity, and diversity of inputs to microbial communities all affect microbial enzyme activity, the mechanisms controlling these relationships are unique for each particular enzyme.     

The mechanism of the phosphoglucomutase reaction. Studies on rabbit muscle phosphoglucomutase with flux techniques

1. The kinetics of phosphoglucomutases from different sources are discussed and it is concluded that on the available evidence there are in all cases three possible mechanisms for the reaction. These are an indirect transfer of phosphate involving the phosphoenzyme (mechanism 1), a direct transfer of phosphate (mechanism 2), and an intermolecular transfer of phosphate from glucose 1,6-diphosphate to the substrate (mechanism 3). Conventional net flux measurements are shown not to differentiate between these mechanisms. 2. Flux equations are developed and it is shown that there are three flux ratios that characterize and distinguish between the mechanisms. 3. To examine these flux ratios induced-transport tests are described with ¹⁴C- and ³²P-labelled substrates. The fluxes determined with ¹⁴C- and ³²P-labelled substrates are also compared at chemical equilibrium. 4. With rabbit muscle phosphoglucomutase the results of these tests were completely consistent with mechanism 1 and unequivocally excluded any substantial part of the reaction proceeding by mechanism 2 or mechanism 3. Evidence was also obtained for an isomerization of the phosphoenzyme with an apparent rate constant about 4·5×10⁷sec.⁻¹. Taking into account the activity coefficients of the substrates the true rate constant appears to be about one-sixth of this value. 5. Isotope effects and non-ideal behaviour of the solutions are discussed and the activity coefficients of the substrates are shown to be equal by measurement of the depression of freezing point. It is concluded that these factors do not influence the tests significantly. 6. Alternative mechanisms are considered and it is concluded that the tests show that the glucose residue is transferred directly, that the phosphate is transferred indirectly with one intermediate phosphate, and that there is an isomerization of the free phosphoenzyme without reference to any other details of the reaction. Further, no assumptions are required about the constancy of rate constants. 7. The relative merits of induced transport and product inhibition for detecting isomerization of the enzyme are discussed. It is concluded that the induced-transport test is more sensitive and that its interpretation is less equivocal. 8. The application of the tests to other enzyme systems is briefly considered.     

Kinetic mechanism of monoamine oxidase A

Steady-state kinetic data for monoamine oxidase A in crude extracts suggest an exclusively ping-pong mechanism, in contrast to those for monoamine oxidase B, which indicate alternate mechanisms involving either a binary or ternary complex. In this study, with use of purified monoamine oxidase A, steady-state data for the inhibition by D-amphetamine of the oxidation of primary amines indicate the possibility of a ternary complex mechanism for monoamine oxidase A also. Stopped-flow studies demonstrate that the rate of reoxidation of reduced enzyme is enhanced by substrates but not by the product, 1-methyl-4-phenylpyridinium. Thus, for the A enzyme, the ternary complex with substrate, but not product, is reoxidized at a faster rate than the free, reduced enzyme. For both the A and B forms of monoamine oxidase, the mechanism is determined by competition between alternate pathways on the basis of the relative rate constants and dissociation constants.