Monitoring the reaction of carbachol with acetylcholinesterase by thioflavin T fluorescence and acetylthiocholine hydrolysis

Acetylcholinesterase (AChE) contains a narrow and deep active site gorge with two sites of ligand binding, an acylation site (or A-site) at the base of the gorge and a peripheral site (or P-site) near the gorge entrance. The P-site contributes to catalytic efficiency by transiently binding substrates on their way to the acylation site, where a short-lived acyl enzyme intermediate is produced. Carbamates are very poor substrates that, like other AChE substrates, form an initial enzyme-substrate complex and proceed to an acylated enzyme intermediate which is then hydrolyzed. However, the hydrolysis of the carbamoylated enzyme is slow enough to resolve the acylation and deacylation steps on the catalytic pathway. Here we show that the reaction of carbachol (carbamoylcholine) with AChE can be monitored both with acetylthiocholine as a reporter substrate and with thioflavin T as a fluorescent reporter group. The fluorescence of thioflavin T is strongly enhanced when it binds to the P-site of AChE, and this fluorescence is partially quenched when a second ligand binds to the A-site to form a ternary complex. These fluorescence changes allow not only the monitoring of the course of the carbamoylation reaction but also the determination of carbachol affinities for the A- and P-sites.     

Transient state kinetic studies of proton liberation by myosin and subfragment 1.

Myosin and subfragment 1 give a maximum burst size of 0.25 to 0.30 protons per active site at pH 8 with ATP, alpha,beta-methylene-ATP, ADP, and adenylyl imidodiphosphate as substrates. The proton is derived from a change in conformation of the enzyme-substrate complex since it is produced by substrates which are not hydrolyzed. The rate constants for the binding of ATP and the proton release step in 0.1 M, 0.5 M, and 1.0 M KCl have been determined by analysis of the concentration dependence of the apparent rate. (see article)     

Oxidation of 4-tert-butylcatechol and dopamine by hydrogen peroxide catalysed by horseradish peroxidase.

The catalytic cycle of horseradish peroxidase (HRP; donor:hydrogen peroxide oxidoreductase; EC 1.11.1.7) is initiated by a rapid oxidation of it by hydrogen peroxide to give an enzyme intermediate, compound I, which reverts to the resting state via two successive single electron transfer reactions from reducing substrate molecules, the first yielding a second enzyme intermediate, compound II. To investigate the mechanism of action of horseradish peroxidase on catechol substrates we have studied the oxidation of both 4-tert-butylcatechol and dopamine catalysed by this enzyme. The different polarity of the side chains of both o-diphenol substrates could help in the understanding of the nature of the rate-limiting step in the oxidation of these substrates by the enzyme. The procedure used is based on the experimental data to the corresponding steady-state equations and permitted evaluation of the more significant individual rate constants involved in the corresponding reaction mechanism. The values obtained for the rate constants for each of the two substrates allow us to conclude that the reaction of horseradish peroxidase compound II with o-diphenols can be visualised as a two-step mechanism in which the first step corresponds to the formation of an enzyme-substrate complex, and the second to the electron transfer from the substrate to the iron atom. The size and hydrophobicity of the substrates control their access to the hydrophobic binding site of horseradish peroxidase, but electron density in the hydroxyl group of C-4 is the most important feature for the electron transfer step.     

Repositioning of the reaction intermediate within the catalytic center of the spliceosome

Conformational change within the spliceosome is required between the first catalytic step of pre-mRNA splicing, when the branch site attacks the 5' splice site (SS), and the second step, when the 5' exon attacks the 3'SS. Little is known, however, about repositioning of the reaction substrates during this transition. Whereas the 5'SS is positioned for the first step by pairing with the invariant U6 snRNA-ACAGAG site, we demonstrate that this pairing interaction must be disrupted to allow transition to the second step. We propose that removal of the branch structure from the catalytic center is in competition with binding of the 3'SS substrate for the second step. Changes in the relative occupancy of first and second step substrates at the catalytic center alter efficiency of the two steps of splicing, allowing use of suboptimal intron sequences and thereby altering substrate selectivity.     

Free energy analysis of ω-transaminase reactions to dissect how the enzyme controls the substrate selectivity

ω-Transaminase (ω-TA) is the only naturally occurring enzyme allowing asymmetric amination of ketones for production of chiral amines. The active site of the enzyme was proposed to consist of two differently sized substrate binding pockets and the stringent steric constraint in the small pocket has presented a significant challenge to production of structurally diverse chiral amines. To provide a mechanistic understanding of how the (S)-specific ω-TA from Paracoccus denitrificans achieves the steric constraint in the small pocket, we developed a free energy analysis enabling quantification of individual contributions of binding and catalytic steps to changes in the total activation energy caused by structural differences in the substrate moiety that is to be accommodated by the small pocket. The analysis exploited kinetic and thermodynamic investigations using structurally similar substrates and the structural differences among substrates were regarded as probes to assess how much relative destabilizations of the reaction intermediates, i.e. the Michaelis complex and the transition state, were induced by the slight change of the substrate moiety inside the small pocket. We found that ≈80% of changes in the total activation energy resulted from changes in the enzyme-substrate binding energy, indicating that substrate selectivity in the small pocket is controlled predominantly by the binding step (K_M) rather than the catalytic step (k_cat). In addition, we examined the pH dependence of the kinetic parameters and the pH profiles of the K_M and k_cat values suggested that key active site residues involved in the binding and catalytic steps are decoupled. Taken together, these findings suggest that the active site residues forming the small pocket are mainly engaged in the binding step but not significantly involved in the catalytic step, which may provide insights into how to design a rational strategy for engineering of the small pocket to relieve the steric constraint toward bulky substituents.     

Kinetic and magnetic resonance studies of substrate binding to galactose oxidase copper(II)

Alcohol substrate binding to the copper-containing enzyme galactose oxidase (GOase) has been studied by kinetic competition against cyanide and fluoride, 13C nmr relaxation, and esr competition experiments. The 13C nmr spectra of the substrate beta-O-methyl-D-galactopyranoside (beta-O-me-gal) show no apparent paramagnetic relaxation rate enhancement that could be attributed to innersphere equatorial binding of this molecule at the Cu(II) center. Moreover, the kinetics observed when CN- or F- are used as inhibitors of GOase with beta-O-me-gal as the substrate suggest that these anions act as apparent non-competitive inhibitors; the binding of the substrates beta-O-me-gal and O2 is not hindered per se, but the catalytic activity of the enzyme substrate complex is greatly decreased. The esr competition data also confirm that, in the absence of O2, CN- and beta-O-me-gal do not compete for the same GOase binding site. Previously reported esr and 19F nmr data show that CN- binds to the GOase Cu(II) at an equatorial coordination site, as does the F- detected in esr experiments. Thus, the results from the various competition experiments supports a model in which alcohol substrates bind outersphere to the GOase Cu(II), or, possibly, to an axial site.     

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.     

Kinetics of the action of papain on fluorescent peptide substrates.

Kinetic measurements have been performed on the action of papain on mansyl-Gly-Val-Glu-Leu-Gly and on mansyl-Gly-Gly-Val-Glu-Leu-Gly, both of which are cleaved solely at the Glu-Leu bond under the conditions of our experiments. Stopped-flow experiments have shown that, under conditions of enzyme excess, the enhancement of the fluorescence of the mansyl group upon association of each of the oligopeptide substrates with papain is a biphasic process. A very rapid initial increase in fluorescence is followed by a slower first-order fluorescence enhancement. The observed rate constant for the latter process is greater with the mansyl pentapeptide than with the mansyl hexapeptide. A similar biphasic fluorescence change is seen upon the interaction of the mansyl peptides with mercuripapain, but the second step is much slower than in the case of the active enzyme. The rate of the second step in the association of active papain with the mansyl paptides shows saturation with increasing enzyme concentration, supporting the view that an initial enzyme-substrate complex (ES) is converted in a first-order process to the complex (ES) that undergoes cleavage to form products. The hydrolysis of the Glu-Leu bond is associated with a first-order decrease in fluorescence, as a consequence of the formation of the mansyl peptide product, which is bound less strongly than the substrate. The rate constant for this process is about 140 times greater with the mansyl hexapeptide than with the mansyl pentapeptide, thus giving further indication of the importance of secondary enzyme-substrate interactions in the efficiency of papain catalysis. For each of the two mansyl peptides, the values of the rate constants and the apparent Michaelis constants associated with the cleavage of the Glu-Leu bond, as determined by stopped-flow measurements under conditions of enzyme excess, were the same, within the precision of the data, as those estimated from experiments under conditions of substrate excess, where the formation of Leu-Gly was determined by means of the fluorescamine reaction. This indicates that, with these substrates, the rate-limiting step in the overall catalytic process is associated with the breakdown of ES. Estimates are given of the dissociation constant of ES and of the rate constants in the interconversion of ES and ES.     

Computational and experimental studies of the catalytic mechanism of Thermobifida fusca cellulase Cel6A (E2)

Mutagenesis experiments suggest that Asp79 in cellulase Cel6A (E2) from Thermobifida fusca has a catalytic role, in spite of the fact that this residue is more than 13 A from the scissile bond in models of the enzyme-substrate complex built upon the crystal structure of the protein. This suggests that there is a substantial conformational shift in the protein upon substrate binding. Molecular mechanics simulations were used to investigate possible alternate conformations of the protein bound to a tetrasaccharide substrate, primarily involving shifts of the loop containing Asp79, and to model the role of water in the active site complex for both the native conformation and alternative low-energy conformations. Several alternative conformations of reasonable energy have been identified, including one in which the overall energy of the enzyme-substrate complex in solution is lower than that of the conformation in the crystal structure. This conformation was found to be stable in molecular dynamics simulations with a cellotetraose substrate and water. In simulations of the substrate complexed with the native protein conformation, the sugar ring in the -1 binding site was observed to make a spontaneous transition from the (4)C(1) conformation to a twist-boat conformer, consistent with generally accepted glycosidase mechanisms. Also, from these simulations Tyr73 and Arg78 were found to have important roles in the active site. Based on the results of these various MD simulations, a new catalytic mechanism is proposed. Using this mechanism, predictions about the effects of changes in Arg78 were made which were confirmed by site-directed mutagenesis.     

Use of binding energy in catalysis analyzed by mutagenesis of the tyrosyl-tRNA synthetase

The utilization of enzyme-substrate binding energy in catalysis has been investigated by experiments on mutant tyrosyl-tRNA synthetases that have been generated by site-directed mutagenesis. The mutants are poorer enzymes because they lack side chains that form hydrogen bonds with ATP and tyrosine during stages of the reaction. The hydrogen bonds are not directly involved in the chemical processes but are at some distance from the seat of reaction. The free energy profiles for the formation of enzyme-bound tyrosyl adenylate and the equilibria between the substrates and products were determined from a combination of pre-steady-state kinetics and equilibrium binding methods. By comparison of the profile of each mutant with wild-type enzyme, a picture is built up of how the course of reaction is affected by the influence of each side chain on the energies of the complexes of the enzyme with substrates, transition states, and intermediates (tyrosyl adenylate). As the activation reaction proceeds, the apparent binding energies of certain side chains with the tyrosine and nucleotide moieties increase, being weakest in the enzyme-substrate complex, stronger in the transition state, and strongest in the enzyme-intermediate complex. Most marked is the interaction of Cys-35 with the 3'-hydroxyl of the ribose. Removal of the side chain of Cys-35 leads to no change in the dissociation constant of ATP but causes a 10-fold lowering of the catalytic rate constant. It contributes no net apparent binding energy in the E X Tyr X ATP complex and stabilizes the transition state by 1.2 kcal/mol and the E X Tyr-AMP complex by 1.6 kcal/mol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hydrolysis of dansyl-peptide substrates by leucine aminopeptidase: origin of dansyl fluorescence changes during hydrolysis

The origin of the fluorescence changes observed in stopped-flow experiments of the hydrolysis of three 5-(dimethylamino)naphthalene-1-sulfonyl-(dansyl) peptide substrates by porcine kidney cytosol leucine aminopeptidase has been investigated. The substrates used all have the potential to accept energy from aromatic residues of the enzyme via resonance energy transfer when they are bound as enzyme-substrate complexes, indicating that fluorescence changes due to the buildup and decay of such intermediates are possible. However, the fluorescence of these substrates differs from that of the products, and direct excitation of their dansyl groups during hydrolysis can also be responsible for the observed fluorescence changes due to changes in the concentrations of free substrate and product. The dansyl fluorescence changes observed with excitation wavelengths near 280 nm are not accompanied by quenching of the enzyme fluorescence, as would be expected if there were enzyme-to-substrate energy transfer. The magnitude of the maximal fluorescence change at a fixed concentration of substrate is also independent of the enzyme concentration. Furthermore, the excitation profile for the fluorescence changes shows that they arise from direct excitation of the dansyl group. Thus, there is no energy transfer in these reactions, and the fluorescence changes observed arise from direct excitation of the dansyl group and reflect the instantaneous concentration of substrate. This behavior contrasts sharply with that for the reaction of carboxypeptidase A with dansyl-Gly-Tyr, which has been studied as a positive control for an energy-transfer system.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phospholipase A2 at the bilayer interface.

Interfacial catalysis is a necessary consequence for all enzymes that act on amphipathic substrates with a strong tendency to form aggregates in aqueous dispersions. In such cases the catalytic event occurs at the interface of the aggregated substrate, the overall turnover at the interface is processive, and it is influenced the molecular organization and dynamics of the interface. Such enzymes can access the substrate only at the interface because the concentration of solitary monomers of the substrate in the aqueous phase is very low. Moreover, the microinterface between the bound enzyme and the organized substrate not only facilitates formation of the enzyme-substrate complex, but a longer residence time of the enzyme at the substrate interface also promotes high catalytic processivity. Binding of the enzyme to the substrate interface as an additional step in the overall catalytic turnover permits adaptation of the Michaelis-Menten formalism as a basis to account for the kinetics of interfacial catalysis. As shown for the action of phospholipase A2 on bilayer vesicles, binding equilibrium has two extreme kinetic consequences. During catalysis in the scooting mode the enzyme does not leave the surface of the vesicle to which it is bound. On the other hand, in the hopping mode the absorption and desorption steps are a part of the catalytic turnover. In this minireview we elaborate on the factors that control binding of pig pancreatic phospholipase A2 to the bilayer interface. Binding of PLA2 to the interface occurs through ionic interactions and is further promoted by hydrophobic interactions which probably occur along a face of the enzyme, with a hydrophobic collar and a ring of cationic residues, through which the catalytic site is accessible to substrate molecules in the bilayer. An enzyme molecule binds to the surface occupied by about 35 lipid molecules with an apparent dissociation constant of less than 0.1 pM for the enzyme on anionic vesicles compared to 10 mM on zwitterionic vesicles. Results at hand also show that aggregation or acylation of the protein is not required for the high affinity binding or catalytic interaction at the interface.     

Effect of modifiers on the hydrolysis of basic and neutral peptides by carboxypeptidase B1.

The initial rates of hydrolysis of Bz-Gly-Lys and Bz-Gly-Phe by carboxypeptidase B (CPB) are increased in the presence of the modifiers beta-phenylpropionic acid, cyclohexanol, Bz-Gly, and Bz-Gly-Gly. The hydrolysis of the tripeptide Bz-Gly-Gly-Phe is also activated by Bz-Gly and Bz-Gly-Gly, but none of these modifiers activate the hydrolysis of Bz-Gly-Gly-Lys, Z-Leu-Ala-Phe, or Bz-Gly-phenyllactic acid by CPB. All modifiers except cyclohexanol display inhibitory modes of binding when present in high concentration. Examination of Lineweaver-Burk plots in the presence of fixed concentrations of Bz-Gly has shown that activation of the hydrolysis of neutral and basic peptides by CPB, as reflected in the values of the extrapolated parameters, Km(app) and kcat, occurs by different mechanisms. For Bz-Gly-Gly-Phe, activation occurs because the enzyme-modifier complex has a higher affinity than the free enzyme for the substrate, whereas activation of the hydrolysis of Bz-Gly-Lys derives from an increase in the rate of breakdown of the enzyme-substrate complex to give products. Cyclohexanol differs from Bz-Gly and Bz-Gly-Gly in that it displays no inhibitory mode of binding with any of the substrates examined, activates only the hydrolysis of dipeptides by CPB, and has a greater effect on the hydrolysis of the basic dipeptide than on the neurtal dipeptide. Moreover, when Bz-Gly-Lys is the substrate, cyclohexanol activates its hydrolysis by CPB by increasing both the enzyme-substrate binding affinity and the rate of the catalytic step, an effect different from that observed when Bz-Gly is the modifier. The anomalous kinetic behavior of CPB is remarkably similar to that of carboxypeptidase A, and is a good indication that both enzymes have very similar structures in and around their respective active sites. A binding site for activator molecules down the cleft of the active site is proposed for CPB to explain the observed kinetic behavior.     

Acquisition of novel catalytic activity by the M1 RNA ribozyme: the cost of molecular adaptation.

The ribonucleoprotein RNase P is a critical component of metabolism in all known organisms. In Escherichia coli, RNase P processes a vast array of substrates, including precursor-tRNAs and precursor 4. 5S RNA. In order to understand how such catalytic versatility is achieved and how novel catalytic activity can be acquired, we evolve the M1 RNA ribozyme (the catalytic component of E. coli RNase P) in vitro for cleavage of a DNA substrate. In so doing, we probe the consequences of enhancing catalytic activity on a novel substrate and investigate the cost this versatile enzyme pays for molecular adaptation. A total of 25 generations of in vitro evolution yield a population showing more than a 1000-fold increase in DNA substrate cleavage efficiency (kcat/KM) relative to wild-type M1 RNA. This enhancement is accompanied by a significant reduction in the ability of evolved ribozymes to process the ptRNA class of substrates but also a contrasting increase in activity on the p4.5S RNA class of substrates. This change in the catalytic versatility of the evolved ribozymes suggests that the acquired activity comes at the cost of substrate versatility, and indicates that E. coli RNase P catalytic flexibility is maintained in vivo by selection for the processing of multiple substrates. M1 RNA derivatives enhance cleavage of the DNA substrate by accelerating the catalytic step (kcat) of DNA cleavage, although overall processing efficiency is offset by reduced substrate binding. The enhanced ability to cleave a DNA substrate cannot be readily traced to any of the predominant mutations found in the evolved population, and must instead be due to multiple sequence changes dispersed throughout the molecule. This conclusion underscores the difficulty of correlating observed mutations with changes in catalytic behavior, even in simple biological catalysts for which three-dimensional models are available.     

The mechanism of p21-activated kinase 2 autoactivation

The p21-activated kinases (PAKs) play an important role in diverse cellular processes. PAK2 is activated by autophosphorylation upon binding of small G proteins such as Cdc42 and Rac in the GTP-bound state. However, the mechanism of PAK2 autophosphorylation in vitro is unclear. In the present study, the kinetic theory of the substrate reaction during modification of enzyme activity has been applied to a study of the autoactivation of PAK2. On the basis of the kinetic equation of the substrate reaction during the autophosphorylation of PAK2, the activation rate constants for the free enzyme and enzyme-substrate complex have been determined. The results indicate that 1) in the presence of Cdc42, PAK2 autophosphorylation is a bipartite mechanism, with the regulatory domain autophosphorylated at multiple residues, whereas activation coincides with autophosphorylation of the catalytic domain at Thr-402; 2) the autophosphorylation reactions in regulatory domain are either a nonlimiting step or not required for activation of enzyme; 3) the autophosphorylation at site Thr-402 on the catalytic domain occurs by an intermolecular mechanism and is required for phosphorylation of exogenous substrates examined; 4) binding of the exogenous protein/peptide substrates at the active site of PAK2 has little or no effect on the autoactivation of PAK2, suggesting that multiple regions of PAK2 are involved in the enzyme-substrate recognition. The present method also provides a novel approach for studying autophosphorylation reactions. Since the experimental conditions used resemble more closely the in vivo situation where the substrate is constantly being turned over while the enzyme is being modified, this new method would be particularly useful when the regulatory mechanisms of the reversible phosphorylation reaction toward certain enzymes are being assessed.     

Kinetic consequences of a slow substrate binding step in group transferases. Interpretation of product inhibition experiments.

The steady state kinetic properties of a simple model for an enzyme catalyzed group transfer reaction between two substrates have been calculated. One substrate is assumed to bind slowly and the other rapidly to the enzyme. Apparent substrate inhibition or substrate activation by the rapidly binding substrate may result if the slowly binding substrate binds at unequal rates to the free enzyme and to the complex between the enzyme and the rapidly binding substrate. Competitive inhibition by each product with respect to its structurally analogous substrate is to be expected if both substrates are in rapid equilibrium with their enzyme-substrate complexes. This product inhibition pattern, however, may also be observed when one substrate binds slowly. Noncompetitive inhibition with respect to the rapidly binding substrate by its structurally analogous product may result if the slowly binding substrate binds more slowly to the enzyme-product complex than to the free enzyme. Inhibition by substrate analogs which are not products should follow the same rules as inhibition by products. Thus substrate analog inhibition experiments are not particularly informative. The form of inhibition by "transition state analog" inhibitors should reveal which substrate binds slowly. There is no sharp conceptual distinction between ordered and random "kinetic mechanisms". I therefore suggest that the use of these concepts should be abandoned.     

Sunflower Seed Aminopeptidase-Catalyzed Hydrolysis of Phe-Xaa Dipeptides — Exploring the S1′ Specificity of the Enzyme

The S₁′ substrate specificity of the sunflower seed major aminopeptidase was studied with a series of dipeptide substrates with phenylalanine at P₁ and a hydrophobic amino acid at P₁′ position. The kinetic parameters of hydrolysis are significantly affected by the structure, side chain hydrophobicity and configuration of the P₁′ moiety. Its binding during enzyme-substrate complex formation takes place at a hydrophobic site of limited size following an extraction mechanism as seen from the applied structure-activity correlation. Attempts to establish such dependencies for the catalytic step of the reaction reveal the presence of additional S₁′-P₁′ enzyme-substrate interactions of greater complexity.     

Conformational change induced by ATP binding in the multidrug ATP-binding cassette transporter BmrA

ATP-binding cassette (ABC) transporters are involved in the transport of a wide variety of substrates, and ATP-driven dimerization of their nucleotide binding domains (NBDs) has been suggested to be one of the most energetic steps of their catalytic cycle. Taking advantage of the propensity of BmrA, a bacterial multidrug resistance ABC transporter, to form stable, highly ordered ring-shaped structures [Chami et al. (2002) J. Mol. Biol. 315, 1075-1085], we show here that addition of ATP in the presence of Mg2+ prevented ring formation or destroyed the previously formed rings. To pinpoint the catalytic step responsible for such an effect, two classes of hydrolysis-deficient mutants were further studied. In contrast to hydrolytically inactive glutamate mutants that behaved essentially as the wild-type, lysine Walker A mutants formed ring-shaped structures even in the presence of ATP-Mg. Although the latter mutants still bound ATP-Mg, and even slowly hydrolyzed it for the K380R mutant, they were most likely unable to undergo a proper NBD dimerization upon ATP-Mg addition. The ATP-driven dimerization step, which was still permitted in glutamate mutants and led to a stable conformation suitable to monitor the growth of 2D crystals, appeared therefore responsible for destabilization of the BmrA ring structures. Our results provide direct visual evidence that the ATP-induced NBD dimerization triggers a conformational change large enough in BmrA to destabilize the rings, which is consistent with the assumption that this step might constitute the "power stroke" for ABC transporters.     

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.