Rat butyrylcholinesterase-catalysed hydrolysis of N-alkyl homologues of benzoylcholine

The purpose of this work was to study the catalytic properties of rat butyrylcholinesterase with benzoylcholine (BzCh) and N-alkyl derivatives of BzCh (BCHn) as substrates. Complex hysteretic behaviour was observed in the approach to steady-state kinetics for each ester. Hysteresis consisted of a long lag phase with damped oscillation. The presence of a long lag phase, with no oscillations, in substrate hydrolysis by rat butyrylcholinesterase was also observed with N-methylindoxyl acetate as substrate. Hysteretic behaviour was explained by the existence of two interconvertible butyrylcholinesterase forms in slow equilibrium, while just one of them is catalytically active. The damped oscillations were explained by the existence of different substrate conformational states and/or aggregates (micelles) in slow equilibrium. Different substrate conformational states were confirmed by 1H-NMR. The K(m) values for substrates decreased as the length of the alkyl chain increased. High affinity of the enzyme for the longest alkyl chain length substrates was explained by multiple hydrophobic interactions of the alkyl chain with amino acid residues lining the active site gorge. Molecular modelling studies supported this interpretation; docking energy decreased as the length of the alkyl chain increased. The long-chain substrates had reduced k(cat) values. Docking studies showed that long-chain substrates were not optimally oriented in the active site for catalysis, thus explaining the slow rate of hydrolysis. The hydrolytic rate of BCH12 and longer alkyl chain esters vs. substrate concentration showed a premature plateau far below V(max). This was due to the loss of substrate availability. The best substrates for rat butyrylcholinesterase were short alkyl homologues, BzCh - BCH4.     

Damped oscillatory hysteretic behaviour of butyrylcholinesterase with benzoylcholine as substrate.

Steady-state kinetics for the hydrolysis of benzoylcholine (BzCh) and benzoylthiocholine (BzSCh) by wild-type human butyrylcholinesterase (BuChE) and by the peripheral anionic site mutant D70G were compared. kcat/Km for the hydrolysis of BzSCh was 17-fold and 32-fold lower than that for hydrolysis of BzCh by wild-type and D70G, respectively. The rate-limiting step for hydrolysis of BzCh was deacylation, whereas acylation was rate-limiting for hydrolysis of BzSCh. Wild-type enzyme and the D70G mutant were found to reach steady-state velocity slowly with BzCh as the substrate. At pH 6, the approach to steady-state for both enzymes consisted of a mono-exponential acceleration upon which a set of damped oscillations was superimposed. From pH 7 to 8.5, the approach to steady-state consisted of a simple exponential acceleration. The damped oscillations were analyzed by both a numerical approximation and simulation based on a theoretical model. BuChE-catalyzed hydrolysis of the thiocholine analogue of BzCh showed neither lags nor oscillations, under the same conditions. The frequency and amplitude of the damped oscillations decreased as the BzCh concentration increased. The apparent induction time for the exponential portion of the lag was calculated from the envelope of the damped oscillations or from the smooth lag. Wild-type BuChE showed a hyperbolic increase in induction time as the BzCh concentration increased (tau max = 210 s at pH 6.0). However, the induction time for D70G was constant over the whole range of BzCh concentrations (tau max = 60 s at pH 6.0). Thus, the induction time does not conform to a simple hysteretic model in which there is a slow conformational transition of the enzyme from an inactive form E to an active form E'. No pH-dependence of the induction time was found between pH 6.0 and 8.5 in sodium phosphate buffers of various concentrations (from 1 mm to 1 m). However, increasing the pH tended to abolish the oscillations (increase the damping factor). This effect was more pronounced for D70G than for wild-type. Although the lyotropic properties of phosphate change from chaotropic at pH 6.0 to kosmotropic at pH > 8.0, no effect of phosphate concentration on the oscillations was noticed at the different pH values, suggesting that the oscillations are not related to a pH-dependent Hofmeister effect of phosphate ions. Simulation and theoretical analysis of the oscillatory behaviour of the approach to the steady-state for BuChE led us to propose a model for the hysteresis of BuChE with BzCh. In this model, the substrate-free enzyme is present as an equilibrium mixture of two forms, E and E'. Substrate binds to E and E', but only Epsilon'S makes products. It is proposed that oscillations originate from a time-dependent change in the local concentration, solvation and/or conformation of substrate in the bulk solution. 1H-NMR measurements provided evidence for a slow equilibrium between two BzCh conformers. Binding of the conformationally preferred substrate conformer leads to products.     

Hysteresis of insect acetylcholinesterase

Pre-steady-state catalytic properties of insect acetylcholinesterase (AChE, EC 3.1.1.7) were studied with the neutral substrate N-methylindoxylacetate. Kinetics of soluble Apis mellifera and Drosophila melanogaster AChE forms showed lags (v(i)=0) before reaching the steady-state. Results were interpreted in terms of slow equilibrium between two conformational states E and E' of insect AChE. Hysteresis of insect AChE has been pointed out for the first time. The hysteretic behaviour was found to depend on the NMIA concentration and the nature of the enzyme. The maximum induction times (tau(max)) to reach the steady-state were 800 and 1000s with soluble AChE from A. mellifera and D.melanogaster, respectively. The orders of magnitude of the tau(max) were high and similar to human AChE and BuChE.     

A theoretical treatment of damped oscillations in the transient state kinetics of single-enzyme reactions

An extension of the available kinetic theory for reactions in the transient state is presented which establishes that single-enzyme reactions may exhibit damped oscillations under the conditions of standard kinetic experiments performed by stopped-flow techniques. Such oscillations may occur for reasonable magnitudes of rate constants in the enzymic reaction mechanism and at physiological concentrations of enzyme and substrate. In the simplest reaction systems, the oscillations will be strongly damped and lead to progress curves resembling those of a reaction governed by standard exponential transients; statistical regression methods may then have to be applied for their detection and characterization. The observation that single-enzyme reactions may exhibit oscillatory behaviour points to a previously unrecognized possible source of the damped oscillations observed in metabolic systems such as the pathways of glycolysis or photosynthesis.     

The dynamics of single-substrate continuous cultures: the role of transport enzymes

A chemostat limited by a single growth-limiting substrate displays a rich spectrum of dynamics. Depending on the flow rate and feed concentration, the chemostat settles into a steady state or executes sustained oscillations. The transients in response to abrupt increases in the flow rate or the feed concentration are also quite complex. For example, if the increase in the flow rate is small, there is no perceptible change in the substrate concentration. If the increase in the flow rate is large, there is a large increase in the substrate concentration lasting several hours or days before the culture adjusts to a new steady state. In the latter case, the substrate concentration and cell density frequently undergo damped oscillations during their approach to the steady state. In this work, we formulate a simple structured model containing the inducible transport enzyme as the key intracellular variable. The model displays the foregoing dynamics under conditions similar to those employed in the experiments. The model suggests that long recovery times (on the order of several hours to several days) can occur because the initial transport enzyme level is too small to cope with the increased substrate supply. The substrate concentration, therefore, increases until the enzyme level is built up to a sufficiently high level by the slow process of enzyme induction. Damped and sustained oscillations can occur because transport enzyme synthesis is autocatalytic, and hence, destabilizing. At low dilution rates, the response of stabilizing processes, such as enzyme dilution and substrate consumption, becomes very slow, leading to damped and sustained oscillations.     

Hysteretic behaviour and GSSG substrate inhibition shown by glutathione reductase from Phycomyces blakesleeanus

Phycomyces blakesleeanus glutathione reductase shows hysteretic behaviour under experimental conditions, when GSSG substrate inhibition is observed. The progress curves for the reaction show an acceleration phase. The degree of hysteresis varied inversely as the enzyme concentration. It increased when GSSG or NADPH concentration increased, whereas the addition of GSH or NADP+ to the initial reaction mixture prevented it from occurring. In addition, hysteresis was dependent on pH, ionic strength and temperature, decreasing as any of these parameters increased. The parallel effects of pH and ionic strength on the GSSG substrate inhibition and hysteretic behaviour suggest a relationship between these two mechanisms. From the overall results reported in this paper, we propose that the hysteretic behaviour shown by Phycomyces glutathione reductase could be due to a process of time-dependent accumulation of reaction products rather than to a slow conformational change.     

pH modulation of transient state kinetics of enzymes. I. Simple theoretical models

The effect of proton concentration on pre-steady-state kinetics has been investigated theoretically for enzyme reactions involving the breaking of one substrate into two products. Even for the simple double-intermediate mechanism the approach to the steady state may exhibit a rather complex kinetics, which is pH-dependent. This process may even exhibit damped oscillations. A change of pH may completely change this transient kinetics and even suppresses the oscillatory regime. A simple method is presented which allows estimation of the values of the rate and ionization constants. This procedure allows one to distinguish the simple double-intermediate mechanism from a more complex process where the 'fast' binding of the substrate induces a 'slow' conformation change of the enzyme.     

Molecular Basis of the General Base Catalysis of an α/β-Hydrolase Catalytic Triad

The serine-histidine-aspartate triad is well known for its covalent, nucleophilic catalysis in a diverse array of enzymatic transformations. Here we show that its nucleophilicity is shielded and its catalytic role is limited to being a specific general base by an open-closed conformational change in the catalysis of (1R,6R)-2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase (or MenH), a typical α/β-hydrolase fold enzyme in the vitamin K biosynthetic pathway. This enzyme is found to adopt an open conformation without a functional triad in its ligand-free form and a closed conformation with a fully functional catalytic triad in the presence of its reaction product. The open-to-closed conformational transition involves movement of half of the α-helical cap domain, which causes extensive structural changes in the α/β-domain and forces the side chain of the triad histidine to adopt an energetically disfavored gauche conformation to form the functional triad. NMR analysis shows that the inactive open conformation without a triad prevails in ligand-free solution and is converted to the closed conformation with a properly formed triad by the reaction product. Mutation of the residues crucial to this open-closed transition either greatly decreases or completely eliminates the enzyme activity, supporting an important catalytic role for the structural change. These findings suggest that the open-closed conformational change tightly couples formation of the catalytic triad to substrate binding to enhance the substrate specificities and simultaneously shield the nucleophilicity of the triad, thus allowing it to expand its catalytic power beyond the nucleophilic catalysis.     

Partial purification and characterization of acetylcholinesterase (AChE) from the estuarine copepod Eurytemora affinis (Poppe)

Oligohaline copepods such as Eurytemora affinis are widespread in estuaries of northwestern Europe. These minute crustaceans are highly sensitive to contamination and thus serve as useful bioindicators for the monitoring of pollutant effects. The use of decreased cholinesterase (ChE) activity as a sublethal biomarker of exposure to neurotoxic compounds supposes that ChE has been defined in copepods. This study reports the partial purification and characterization of ChE extracted from E. affinis. Analysis by non-denaturing PAGE and by isoelectric focusing indicated that the enzyme is probably a single dimeric form of 140 KDa, with a pI of 6.2. This enzyme is likely an acetylcholinesterase (AChE) since it hydrolyzes acetylthiocholine iodide at a higher rate than other substrates, such as butyrylthiocholine and propionylthiocholine, at pH 7.0 and 25 degrees C, and is inhibited by eserine but not by iso-OMPA. The enzyme exhibited high sensitivity to some of the various pollutants tested. The kinetic properties of this ChE were compared with those of other invertebrate ChEs.     

Expression of cholinesterase gene(s) in human brain tissues: translational evidence for multiple mRNA species

To resolve the origin(s) of the molecular heterogeneity of human nervous system cholinesterases (ChEs), we used Xenopus oocytes, which produce biologically active ChE when microinjected with unfractionated brain mRNA. The RNA was prepared from primary gliomas, meningiomas and embryonic brain, each of which expresses ChE activity with distinct substrate specificities and molecular forms. Sucrose gradient fractionation of DMSO-denatured mRNA from these sources revealed three size classes of ChE-inducing mRNAs, sedimenting at approximately 32S, 20S and 9S. The amounts of these different classes of ChE-inducing mRNAs varied between the three tissue sources examined. To distinguish between ChEs produced in oocytes and having different substrate specificities, their activity was determined in the presence of selective inhibitors. Both 'true' (acetylcholine hydrolase, EC 3.1.1.7) and 'pseudo' (acylcholine acylhydrolase, EC 3.1.1.8) multimeric cholinesterase activities were found in the mRNA-injected oocytes. Moreover, human brain mRNAs inducing 'true' and 'pseudo' ChE activities had different size distribution, indicating that different mRNAs might be translated into various types of ChEs. These findings imply that the heterogeneity of ChEs in the human nervous system is not limited to the post-translational level, but extends to the level of mRNA.     

Nonlinear oscillatory reaction of catalase induced by gradual entry of substrate

Characteristic oscillatory reactions were observed when hydrogen peroxide migrated through semipermeable membrane into a solution of catalase. Measurements were made with DO and an mV meter. Oscillation clearly occurred in the range between 25 degrees C and 37 degrees C and between pH 6.0 and pH 7.6. It was also shown that a driving force for the permeation of H2O2, which was the cause of the oscillations, was a deviation from its equilibrium concentration. We made simulations for oscillatory reactions on the basis of these findings. The result indicated that considering the evaporation of O2 was necessary in order to interpret the oscillatory reactions of catalase in addition to the slow entry of substrate caused by deviation from the equilibrium concentration. The fact that oscillations arise by using this method may provide an important insight into the study of enzyme reactions mediated by membranes in living systems, because many enzyme reactions take place with the mediation of a biomembrane.     

Kinetic studies of the lipid-activated pyruvate oxidase flavoprotein of Escherichia coli

Pyruvate oxidase is a flavoprotein dehydrogenase isolated from Escherichia coli which catalyzes the oxidative decarboxylation of pyruvate to acetate and CO2. In vivo, the enzyme can bind to the bacterial membrane and reduce ubiquinone-8, feeding electrons into the respiratory chain. The purified enzyme has been shown previously to bind to phospholipids and detergents and, upon doing so, is activated. The turnover with ferricyanide as an electron acceptor increases 20- to 30-fold upon lipid binding. In this work, initial velocity and stop-flow kinetics are used to investigate the activation of this enzyme. It is shown that the unactivated form of the enzyme is markedly hysteretic. Progress curves at low substrate concentrations show an initial acceleration in enzyme turnover. This is consistent with the results of stop-flow experiments. Rates obtained for either the reduction of the unactivated flavoprotein by pyruvate or its reoxidation by ferricyanide in single turnover experiments are much slower than the rates predicted by observed turnover in initial velocity studies, in some cases by more than 2 orders of magnitude. The data are best explained by the slow interconversion between two forms of the enzyme, one with low turnover and one which rapidly turns over. As isolated, the enzyme is highly unreactive, as revealed by the stop-flow experiments. During turnover, even in the absence of lipid activators, some of the enzyme converts to the rapid-turnover form. This slow interconversion is shown by kinetic simulation to preclude a steady state from being established. Lipid activators appear to shift the equilibrium to favor the rapid-turnover form of the enzyme. Once the enzyme is "locked" into an activated conformation, the hysteresis is no longer observed, and the stop-flow results are in agreement with data obtained from initial velocity experiments. Activation appears to result in both increased rates of electron transfer into and out of the flavin.     

Modeling of Hysteresis in Gene Regulatory Networks

Hysteresis, observed in many gene regulatory networks, has a pivotal impact on biological systems, which enhances the robustness of cell functions. In this paper, a general model is proposed to describe the hysteretic gene regulatory network by combining the hysteresis component and the transient dynamics. The Bouc-Wen hysteresis model is modified to describe the hysteresis component in the mammalian gene regulatory networks. Rigorous mathematical analysis on the dynamical properties of the model is presented to ensure the bounded-input-bounded-output (BIBO) stability and demonstrates that the original Bouc-Wen model can only generate a clockwise hysteresis loop while the modified model can describe both clockwise and counter clockwise hysteresis loops. Simulation studies have shown that the hysteresis loops from our model are consistent with the experimental observations in three mammalian gene regulatory networks and two E.coli gene regulatory networks, which demonstrate the ability and accuracy of the mathematical model to emulate natural gene expression behavior with hysteresis. A comparison study has also been conducted to show that this model fits the experiment data significantly better than previous ones in the literature. The successful modeling of the hysteresis in all the five hysteretic gene regulatory networks suggests that the new model has the potential to be a unified framework for modeling hysteresis in gene regulatory networks and provide better understanding of the general mechanism that drives the hysteretic function.     

Escherichia coli phosphoenolpyruvate carboxylase: studies on the mechanism of multiple allosteric interactions.

Some kinetic studies of the interactions between Escherichia coli phosphoenolpyruvate carboxylase (orthophosphate:oxaloacetate carboxylase (phosphorylating) EC 4.1.1.31) acetyl coenzyme A, fructose 1,6-bisphosphate, and aspartate were performed. Activation of the enzyme by fructose 1,6-bisphosphate is anomalous by comparison with acetyl coenzyme A in that it confers hysteretic properties on the enzyme. In the presence of both activators and aspartate, hysteresis is observed also, but the approach to optimum catalytic activity can be fit to an equation for a second-order reaction with respect to enzyme concentration. Since, however, hysteresis is not a result of any apparent association-dissociation reaction, the apparent fit to a second-order kinetic equation is probably not real but is the result of a multistep activation mechanism. Hysteresis is not eliminated by preincubation of the enzyme with fructose 1,6-bisphosphate, acetyl coenzyme A, or phosphoenolpyruvate singly or in any pair of combinations. Hysteresis is associated, therefore, with the slow conformation change from the inactive species to the active species under the influence of all three of those reactants. The enzyme complex resulting from the binding of each activator, including phosphoenolpyruvate, has an increased affinity for the other activators. A kinetic method for estimating the relative changes in affinity of these complexes for some of the other reactants is presented. At concentrations of the activators below their K_a, synergistic effects are evident, particularly in their ability to relieve aspartate inhibition. Aspartate inhibition is competitive with acetyl coenzyme A both in the absence and in the presence of low concentrations of fructose 1,6-bisphosphate. Increasing the concentrations of fructose 1,6-bisphosphate results in an increase in the apparent K_l for aspartate, suggesting that synergistic activation by fructose 1,6-bisphosphate is a result of the increased affinity of the fructose 1,6-bisphosphate-enzyme complex for acetyl coenzyme A, and a shift in the concentration of enzyme species away from the one(s) to which aspartate can bind most easily. In the presence of fructose 1,6-bisphosphate alone optimal activation can be achieved, but the concentrations required in vitro are high and suggest that fructose 1,6-bisphosphate alone does not function in that capacity physiologically, but primes the enzyme for more effective activation by acetyl coenzyme A and/or phosphoenolpyruvate.     

Metrifonate induces cholinesterase inhibition exclusively via slow release of dichlorvos

Metrifonate, a long-acting cholinesterase (ChE) inhibitor with very low toxicity in warm-blooded animals, inhibits rat brain and serum cholinesterase (ChE) in vitro through its hydrolytic degradation product, dichlorvos. This conclusion is based on the finding that metrifonate-induced ChE inhibition showed the same pH dependence as its reported dehydrochlorination to dichlorvos. The ChE inhibition induced by dichlorvos was not pH dependent. It was mediated by a competitive drug interaction with the catalytic site of the enzyme, which led to irreversible inhibition within several minutes of incubation. After this time, addition of further substrate to the inhibited enzyme was not able to promote drug dissociation and hence enzyme reactivation. Similar characteristics of inhibition, i.e. interaction with the substrate binding site and time-dependent switch to non-competitive inhibition were observed with the reference compound, physostigmine. However, the physostigmine-induced inhibition of ChE could be readily reversed by further substrate addition. Another reference compound, tetrahydroaminoacridine (THA), also induced a reversible inhibition of rat brain and serum cholinesterase, but with a mechanism of action different from that of both dichlorvos and physostigmine in that enzyme inhibition occurred rapidly upon drug addition at an allosteric site on the enzyme surface. It is suggested that the unique slow release plus the slow inhibition of ChE by dichlorvos is responsible for the lower toxicity of metrifonate compared to that of directly acting ChE inhibitors.     

How the various substrates activate the process of enzymatic hydrolysis by different cholinesterases

Kinetic analysis of the activating effect of substrate on the cholinesterase catalysis is performed. There are determined values of coefficient of activation A in the pH zone 5.0-7.5 for the process of hydrolysis of acetylcholine, indophenylacetate (IPA), and 2,6-dichlorophenolindophenylacetate (DIPA) by cholinesterase (ChE) of horse blood serum, as well as of IPA and DIPA by ChE of optical ganglia of the Pacific squid Todarodes pacificus. The phenomenon of activation has not been revealed at hydrolysis of phenylacetate by the horse blood serum ChE. The conclusion is made that the cause of the activating effect of substrate on the process of enzymatic hydrolysis by ChEs of different origin is the presence of the onium grouping in the structure of substrates.     

Hysteresis,oscillations, and pattern formation in realistic immobilized enzyme systems

Summary Hysteresis, oscillations, and pattern formation in realistic biochemical systems governed by P.D.E.s are considered from both numerical and mathematical points of view. Analysis of multiple steady states in the case of hysteresis, and bifurcation theory in the cases of oscillations and pattern formation, account for the observed numerical results. The possibility to realize these systems experimentally is their main interest, thus bringing further arguments in favor of theories explaining basic biological phenomena by diffusion and reaction.     

pH modulation of transient state kinetics of enzymes. II. Transient state kinetics of plant cell wall acid phosphatase

The pre-steady-state kinetics of plant cell wall acid phosphatase has been investigated at different pH values. The approach of the steady stale lasts about 1 or 2 s and may be fitted with two exponential terms. For certain pH values the approach to the steady state exhibits damped oscillations. Plotting the sum and the product of the two time constants of these exponentials as a function of substrate concentration yields two straight lines. From the slopes and intercepts of these lines one may determine the values of rate and ionization constants involved in the reaction scheme. The results obtained are consistent with the view that the binding of the substrate to the enzyme does not induce a 'slow' conformation change of the enzyme. The enzyme reacts with its substrate while being mostly in its ionized form. Release of p-nitrophenol is also favoured by this ionized form of the enzyme. However, the hydrolysis of the phosphoryl-enzyme complex mostly occurs from the protonated form of the enzyme. The ionization constants of the free enzyme and of the various enzyme-ligand complexes are very similar.     

Bradyrhizobium japonicum isocitrate dehydrogenase exhibits calcium-dependent hysteresis

Bradyrhizobium japonicum NADP(+)-dependent isocitrate dehydrogenase was purified both from cultured cells and from the symbiotic form of the bacteria and was found to be identical in terms of N-terminal amino acid sequence, kinetics, and physicochemical properties. Magnesium and glycerol were absolute requirements for maintaining enzyme activity. The N-terminal amino acid sequence of the enzyme was more similar to the sequences from soybean and yeast than to other bacterial sequences. There was no immunological cross-reaction of antibodies from B. japonicum isocitrate dehydrogenase to extracts of soybean, pea, or Escherichia coli, but there was detectable, although weak, cross-reaction of antibodies from E. coli with the B. japonicum enzyme. B. japonicum isocitrate dehydrogenase displayed strong inhibition by NADH, indicating that during symbiotic nitrogen fixation the enzyme activity would be markedly reduced in planta. The enzyme displayed a calcium-dependent hysteresis, with a pronounced lag lasting as long as 2 min. Hysteresis was evident at concentrations of magnesium less than 0.5 mM and calcium greater than 1 microM. The hysteresis could be alleviated by excess magnesium or by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. The results suggest two roles for magnesium during catalysis; one magnesium may be needed to convert the enzyme into the steady-state form and the second needed for chelation of isocitrate for catalysis. The calcium-dependent hysteretic behavior of B. japonicum NADP(+)-isocitrate dehydrogenase suggested that this metal could serve as an intracellular regulator during symbiosis.     

Hysteretic enzyme response induced by inhibitory antibodies against human leukocyte elastase

Polyclonal antibodies raised in rabbits against human leukocyte elastase contained two distinct populations of enzyme-inhibiting immunoglobulins. The enzyme-catalyzed reaction in the presence of antibodies (both IgG or monovalent Fab fragments) showed a transient state lasting up to several minutes depending on the inhibitor and substrate concentrations, which was followed by a linear steady-state. The transient was a concave upward or concave downward lag phase depending on whether the enzyme had been preincubated with the antibodies or not, respectively. The kinetic analysis of reaction progress curves showed that both antibody populations were slow inhibitors, which completely and reversibly excluded the substrate from binding to the enzyme. For both antibody populations, the formation of the enzyme-inhibitor complex was characterized by an initial rapid interaction followed by a slow isomerization to a catalytically inactive complex. The apparent pseudo first-order rate constant of the transient slow phase was a hyperbolic function of the inhibitor concentration for both antibodies, from which relevant kinetic constants and the half times for enzyme inactivation could be calculated. For instance, with a total antibody concentration of 1 mg/ml (as IgG), leukocyte elastase was inactivated with t1/2 = 0.31s and 24.8s by the faster and the slower of the two antibodies, respectively. It is suggested that the hysteretic response of the enzyme to the inhibitory action of its antibodies may be due to a kind of memory of the antibody molecule for a special inactive enzyme conformation resulting from inhibition by proteinase inhibitors during the immunization procedure. In turn, the purified antibodies would be able to reversibly induce a slow transition of the enzyme molecule from an active to a substrate-excluding conformation ("induced misfit").