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.

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.

Hydrolysis of emulsified mixtures of triacylglycerols by pancreatic lipase.

Hydrolysis of the emulsified mixture of short-chain triacylglycerols by porcine pancreatic lipase in the presence of procolipase and micellar sodium taurodeoxycholate has been studied. Increase in the content of tributyrin and trioctanoin in the mixture with triacetin had highly cooperative effects on the formation of the interfacial lipase procolipase complex. Abrupt enhancement of the complex stability was observed in the presence of 0.4-0.6 mol mol-1 of tributyrin or 0.58 mol mol-1 of trioctanoin in the substrate phase. The affinity of lipase towards interfacially bound procolipase for the trioctanoin containing 0.07-0.42 mol mol-1 of triacetin was approximately three times higher than that for pure trioctanoin. The cooperative processes involved in complex formation did not contribute to the affinity of the interfacial lipase/(pro)colipase complex towards substrate molecules and its catalytic activity.

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.

Aminolysis of acyl-chymotrypsins by amino acids. Kinetic appearance of concentration effect in peptide yield enhancement by freezing.

The effects of reagent concentrations, various added substances, pH and temperature on the yield of peptide synthesis by chymotrypsin in frozen and liquid solutions at subzero temperatures have been studied. Increased nucleophile concentration in the liquid microinclusions of ice has been shown to be sufficient for explaining the peptide yield improvement found at freezing conditions.

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.

Peptide synthesis by chymotrypsin in frozen solutions. Free amino acids as nucleophiles.

Nucleophilic efficiency of the free amino acids in chymotrypsin-catalyzed acyl transfer in ice at -18 degrees C using ethyl esters of N-maleyl-L-tyrosine and L-tyrosine as the acyl group donors has been studied. Although the amino acids did not act as acyl acceptors in liquid water, the high yields of peptides were obtained in frozen solutions at pH 10.5 (before freezing). The efficiency of amino acids in the formation of the corresponding dipeptides depended on the substrate used, and decreased in the order Ser,Thr,Gln > Lys > Cit > Ala > Ala > Gly > Asn > Arg > Glu > Val > Orn > Asp with no peptide formed with His, Leu, Ile and Pro) for N-maleyl-L-tyrosine ethyl ester and Ser > Lys > Orn > Arg,Cit > Gln > Thr > Asn > Ala > Gly (with no peptide formed with Glu, Val, Asp, His, Leu, Ile and Pro) for L-tyrosine ethyl ester.

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.

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.