pH-dependent salting-out effect in enzyme-catalyzed reaction kinetics.

The influence of KCl on the second-order rate constant kII = k2/Ks of acetylcholinesterase-catalyzed hydrolysis of butyl acetate is quantitatively described by the linear equation log kII = log k0II + delta kappa c, where c is the salt concentration and delta kappa = kappa E - kappa X* + kappa s is the difference between the salting-out coefficients of the initial reagents (E and S) and of the transition state (X*). The salting-out parameter delta kappa increases with increasing pH (pH range 4.8-6.0) up to the constant value at pH 6.0-8.0, where delta kappa = kappa s. The pH-dependent delta kappa is discussed as an indication of the change in the volume and/or solvation of the enzyme upon the formation of the transition state. The data show that while the pKa1 = 5.6 +/- 0.1 and pKa2 = 8.0 +/- 0.1 of kII are independent of the KCl concentration, the apparent pKa shift can be observed at high salt concentrations due to a pH-dependent salt effect.

Comparison of salt effects on the reactions of acetylcholinesterase with cationic and anionic inhibitors.

The influence of inorganic salts on the inhibition of acetylcholinesterase by charged organophosphorous inhibitors has been studied. It has been shown that the salt effect on the reaction of acetylcholinesterase with anionic bis(p-nitrophenyl) phosphate is determined by the influence of added salts on the activity coefficient of the inhibitor. In contrast to the salt effects on the reaction of acetylcholinesterase with cationic compounds, it does not include contribution from the enzyme charges. The smaller salt effect in the case of anionic inhibitor can be explained assuming that the anionic inhibitor does not form a non-covalent complex with the enzyme before the phosphorylation step of the reaction. Comparison of salt effects on the substrate turnover showed that in the case of cholinesterases from natural sources they are larger than in the case of enzymes expressed in recombinant cell clones. The enhanced salt effects may result from post-translational modification of the enzyme.

Role of ionic interactions in cholinesterase catalysis.

Acetylcholinesterase (AChE, EC 3.1.1.7) is an enzyme terminating the transmission of nerve impulse in synapses by rapid and selective hydrolysis of the neurotransmitter acetylcholine. Recent years have added a considerable amount of structural knowledge about this protein as well as opened new perspectives to the study of the molecular mechanism of cholinesterase catalysis. In this paper the current state of understanding the molecular recognition by cholinesterases is critically surveyed with particular emphasis on the role of electrostatic interactions.

Acetylcholinesterase as polyelectrolyte: interaction with multivalent cationic inhibitors.

Influence of inorganic salts on the interaction of cobra venom acetylcholinesterase (EC 3.1.1.7) with hexamethonium and gallamine has been studied. The observed negative electrostatic salt effect in the dissociation constant of the enzyme-ligand complex, KD, has been described by equation pKD = pKD degrees-ZL psi +Z log[Me+Z] following from Manning's polyelectrolyte theory, where psi +Z is the fraction of condensed counterions Me+Z per one negative charge of the polyanionic enzyme. The ZL psi+Z values for the complex formation between native acetylcholinesterase and hexamethonium (ZL = +2) or gallamine (ZL = +3) were in quantitative agreement with those predicted by the theory making use of psi+1 = 0.50 found earlier from the influence of salts upon the hydrolysis of acetylcholine by the enzyme. Increase in the number of negative charges in acetylcholinesterase by its modification with pyromellitic dianhydride resulted in an increase of psi+1 to 0.6. The data show that the influence of salts on the electrostatic contribution to the energy of binding of cationic substrates and inhibitors by acetylcholinesterase can be quantitatively described proceeding from the counterion condensation model of Manning by using only one empirical parameter psi+1 for a given subtype or modified form of the enzyme.

Measurement and modelling of sequence-specific pKa values of lysine residues in calbindin D9k.

A pH titration study of calbindin D9k was performed using heteronuclear 1H-13C two-dimensional NMR spectroscopy. The protein was produced with carbon-13 label in the side-chain of lysine residues, next to the titrating group. The site-specific pKa values of these lysine residues, ranging from 10.1 to 12.1, were obtained from the analysis of pH-dependent chemical shifts of 13C and 1H resonances. Ionization constants for both the Ca(2+)-free (apo) and Ca(2+)-loaded forms of the protein were determined. The proton uptake by lysine residues in the apo form was shifted up to 1.7 units towards high pH as compared to that for the model compound. The binding of calcium affected the pKa values of all lysine residues. The largest reduction of one pK unit was observed for Lys55, which is also the closest to the calcium binding sites. A threefold increase in protein concentration, from 0.5 to 1.5 mM, reduced the pKa values by 0.1 to 0.4 pK unit in agreement with the screening concept of ionic interactions. All the observed pKa shifts were site-specific, depending on the local electrostatic environment and were reproduced in Monte Carlo simulations based on the three-dimensional structure of calbindin D9k and a dielectric continuum model for the electrostatic interactions.

Acetylcholinesterase as polyelectrolyte in reaction with cationic substrates.

It is shown that the salt effect in acetylcholinesterase-catalyzed hydrolysis of 2-(N-methylmorpholinium)-ethylacetate can be quantitatively described by the equation log(k2/KS) = log(k2/KS) degrees--psi log[M+Z] following from Manning's polyelectrolyte theory; the psi values for salts with univalent and bivalent cations at different pH values of the reaction medium were in accordance with the conclusions of the theory. Manning's polyelectrolyte theory seems to be a useful framework for studying salt effects in the reactions of charged substrates with enzymes as globular polyions.

Structure-activity relationships in acetylcholinesterase reactions. Hydrolysis of non-ionic acetic esters.

The Michaelis-Menten parameters kcat, Ks(app) and the second-order rate constants kII = k2/Ks of acetylcholinesterase-catalyzed hydrolysis of 25 acetic esters with non-ionic leaving groups have been determined at 25 degree C and pH 7.5 in 0.15 M KCL. A linear relationship between the substrate noncovalent binding capacity and the leaving group hydrophobicity, and a multiparameter correlation of the acetylation reaction rate constant logarithm with the leaving group inductive effect, hydrophobicity, and steric effect, have been established. The acetyl-enzyme deacetylation rate constant has been calculated. Taken together, a fairly complete understanding of acetylcholinesterase specificity is possible. The data are consistent with a model of the acetylcholinesterase active site, in which the catalytically active groups are located at the bottom of a jaws-like slit with a limited range of hydrophobic walls that provide the sorption of the substrate leaving groups not longer than that in n-butyl acetate.

Binding of Ca2+ to calbindin D9k: structural stability and function at high salt concentration.

Calcium binding constants of wild-type calbindin D9k and mutant forms with one, two, and three neutralized negative charges in the vicinity of the Ca2+ binding sites are determined at varying KCl concentrations from 2 mM to 1 M. The results indicate that the added salt does not cause significant structural changes in calbindin D9k and, along with site-directed mutagenesis, can be used as a well-controlled means for modulating electrostatic interactions. The lack of structural changes at high salt concentrations is also supported by two-dimensional 1H NMR data. High salt concentrations are observed to substantially reduce the cooperativity of calcium binding to calbindin D9k. This suggests that the cooperativity is strongly dependent on electrostatic interactions. The data have been used to test a dielectric continuum model for protein electrostatics using a macroscopic dielectric constant of water throughout the system. Excellent agreement between experiment and Monte Carlo simulations is observed for the whole set of data covering changes in the binding constant of more than 6 orders of magnitude. A simplified theoretical treatment using the Kirkwood-Tanford formula, based on the Debye-Hückel approximation, yields an almost equally good agreement with the experiment.

Focusing of the electrostatic potential at EF-hands of calbindin D(9k): titration of acidic residues.

Biological functions for a large class of calmodulin-related proteins, such as target protein activation and Ca(2+) buffering, are based on fine-tuned binding and release of Ca(2+) ions by pairs of coupled EF-hand metal binding sites. These are abundantly filled with acidic residues of so far unknown ionization characteristics, but assumed to be essential for protein function in their ionized forms. Here we describe the measurement and modeling of pK(a) values for all aspartic and glutamic acid residues in apo calbindin D(9k), a representative of calmodulin-related proteins. We point out that while all the acidic residues are ionized predominantly at neutral pH, the onset of proton uptake by Ca(2+) ligands with high pK(a) under these conditions may have functional implications. We also show that the negative electrostatic potential is focused at the bidental Ca(2+) ligand of each site, and that the potential is significantly more negative at the N-terminal binding site.

[Inhibition of acetylcholinesterase hydrolysis of cationic substrates by the reaction product]. / Ingibirovanie atsetilkholinesterazy produktami gidroliza kationnykh substratov.

The kinetics of acetylcholinesterase-catalyzed hydrolysis of the two cationic substrates (I and II in Russian text) was analyzed by means of the integrated Michaelis equation (3). The constants kII, kcat Km and the enzyme-product complex dissociation constant Ki were determined. (Table 1). It was shown that acetylcholine (II) binds to to the enzyme active center more effectively than the alcohol product of its hydrolysis. In case of the pipecholine derivative (I) reversed situation occurs. The different dependence of the ester substrate and appropriate alcohol binding effectiveness upon the reagent structure indicates the dissimilar location of the molecules in the active center of acetylcholinesterase. Some structural implications of the enzyme active center were discussed.

Ionization behavior of acidic residues in calbindin D(9k).

The ionization state of seven glutamate residues, one aspartate, and the C-terminal alpha-COOH group in bovine apo calbindin D(9k) has been studied by measurement and modeling of the pH titration curves and apparent pK(a) values. The observed pK(a) ranged from 3.0 to 6.5. Most of the observed acidic groups were half-ionized at lower pH values than those in unstructured proteins. As a rule, the ionization equilibria extended over a wider pH range than in the case of unperturbed single titrations, indicating a complex influence of protein charges on the charge state of each individual residue. Glu17, which is a backbone Ca(2+)-ligand in the N-terminal binding loop of calbindin D(9k), was half-protonated at pH 3.6 but manifested biphasic titration with apparent pK(a) values of 3.2 and 6.5. Complementary Monte Carlo simulations of the titration process and pK(a) values of the acidic groups in calbindin D(9k) reproduce the experimentally observed titration features, except for the pronounced double titration of Glu17. Discrepancies between the results from direct measurement and from modeling may be partly caused by changes in the protein structure when the net charge changes from -8 to +11 over the isoelectric point at pH 5. Proteins 1999;37:106-115.

Electrostatic effects in trypsin reactions. Influence of salts.

The influence of inorganic salts on trypsin-catalyzed reactions has been studied. It is shown that: (a) monovalent cations are reversible competitive inhibitors of tryptic hydrolysis of cationic substrates, whereas their binding has no effect on the reaction of neutral substrates; (b) a nonelectrostatic salt effect on the binding of both cationic and non-ionic substrates is caused by changes in the thermodynamic activity coefficient of the substrate; (c) the rate of trypsin active-site acylation is not affected by inorganic salts with monovalent cations. The data suggest that low-molecular-mass substrates are extracted into the enzyme microphase during substrate binding and further chemical transformations proceed without an access from surrounding medium. It is proposed that formation of a properly oriented dipole in the trypsin binding pocket by the cationic group of the substrate and Asp189 carboxyl is responsible for the elevated acylation rate of trypsin active site by substrates containing lysine and arginine. Introduction of additional negative charges into the enzyme molecule by chemical modification of lysyl residues by pyromellitic anhydride increased the specificity of trypsin towards cationic substrates and inhibitors. Lysine residues are therefore considered as suitable targets for site-directed mutagenesis aimed at the improvement of selectivity and catalytic properties of trypsin.

Calbindin D(9k): a protein optimized for calcium binding at neutral pH.

The binding of calcium ions by EF-hand proteins depends strongly on the electrostatic interactions between Ca(2+) ions and negatively charged residues of these proteins. We have investigated the pH dependence of the binding of Ca(2+) ions by calbindin D(9k). This protein offers a unique possibility for interpretation of such data since the pK(a) values of all ionizable groups are known. The binding is independent of pH between 7 and 9, where maximum calcium affinity is observed. An abrupt decrease in the binding affinity is observed at pH values below 7. This decrease is due to protonation of acidic groups, leading to modification of protein charges. The pH dependence of the product of the two macroscopic Ca(2+)-binding constants can be formally described by the involvement of two acidic groups with pK(a) = 6.6. Monte Carlo calculations show that the reduction of Ca(2+) binding is strictly determined by variable electrostatic interactions due to pH-dependent changes not only in the binding sites, but also of the overall charge of the protein.

Control of cellular respiration in vivo by mitochondrial outer membrane and by creatine kinase. A new speculative hypothesis: possible involvement of mitochondrial-cytoskeleton interactions.

The current problems of regulation of myocardial energy metabolism and oxidative phosphorylation in vivo are considered. With this purpose, retarded diffusion of ADP in cardiomyocytes was studied by analysis of elevated apparent Km for this substrate in regulation of respiration of saponin-skinned cardiac fibers, as compared to isolated mitochondria. Recently published data showing the importance of the outer mitochondrial membrane were compared with new experimental results on the proteolysis of skinned fibers and tissue homogenates. In both cases 10 min incubation and 0.125 mg/ml of trypsin resulted in a decrease of apparent Km for ADP from 297 +/- 35 and 228 +/- 16 to 109 +/- 2 and 36 +/- 16, respectively. Thus, the permeability of the outer mitochondrial membrane for ADP may be controlled by some unknown cytoplasmic protein(s), probably related to the cytoskeleton, which are separated from mitochondria during their isolation. The extent of expression of this protein(s) depends on the energy state and type of muscle. Activation of mitochondrial creatine kinase reaction coupled to oxidative phosphorylation overcomes the diffusion difficulties of ADP by amplifying the stimulatory effect of ADP on respiration. It is concluded that both cytoplasmic and mitochondrial creatine kinases, adenylate kinase and cytoplasmic factor controlling outer membrane permeability may participate in metabolic feedback regulation of respiration in muscle cells.