Yechiel Shalitin

Immobilization of enzymes by covalent binding to amine supports via cyanogen bromide activation

Abstract Enzymes can be readily linked to amine carrying supports activated by cyanogen bromide. High yields of binding and activity were obtained, and the bound enzymes exhibited greater stability than the free form toward denaturing effectors. In contrast with proteins bound to cyanogen bromide activated Sepharose, those bound to amine supports were not released from the matrix even in the presence of high concentrations of nucleophiles.

2-Aminoperimidine, a specific inhibitor of bacterial NhaA Na+/H+ antiporters

The diuretic drug amiloride and its numerous derivatives are competitive inhibitors of mammalian Na+/H+ antiporters and other eukaryotic antiporters. Most prokaryotic antiporters, including the major NhaA family of enterobacteria, are resistant to these compounds. We show that 2‐aminoperimidine (AP), a guanidine‐containing naphthalene derivative with some similarity to amiloride, acts as a specific inhibitor of NhaA from Escherichia coli. Similar concentrations (IC50 of 0.9 μM) inhibit the proton motive force dependent Na+(Li+)/H+ exchange reaction in inside‐out sub‐bacterial vesicles (at 10 mM NaCl, pH 8) as well as the initial rate of 22Na+/Na+ exchange mediated by pure NhaA in proteoliposomes. The inhibitor is specific to NhaA type antiporters, so AP is a new tool to study the mechanism and roles of NhaA antiporters of enterobacteria as well as the molecular basis of inhibition by an amiloride‐like compound.

New substrates of acetylcholinesterase

Volume 134, number 1 FEBS LETTERS November 1981 NEW SUBSTRATES OF ACETYLCHOLINESTERASE Meira NAVEH, Zmira BERNSTEIN, Dina SEGAL and Yechiel SHALITIN* Department of Biology, Technion-Israel Institute of Technology, Haifa, 32000, Israel Received 2 September 1981 1. Introduction Acetylcholinesterase (ACHE) has been the subject of intensive studies in the last 4 decades [1,2]. Occu- pying a central role in transmission of nervous signals and being an efficient hydrolase model enzyme, AChE attracted much interest, and a vast number of compounds were studied as substrates and inhibitors of the enzyme, for both theoretical and applied rea- sons. Our aim was to examine the ability of new classes of compounds to serve as AChE substrates. A variety of compounds were tested as possible substrates of the Electrophorus enzyme and several highly active substrates were found: (1) Phenothiazine carbonyl chloride was found to be a potent covalent inhibitor of acetylcholinesterase and could be used to titrate the enzyme active site ; * To whom correspondence should be addressed (2) Enol acetates (vinyl acetate and its derivatives) are good substrates of the enzyme, yielding ace- tate and aldehyde upon hydrolysis; (3) Alkylidenediacetates are hydrolyzed by acetyl- cholinesterase, yielding also acetate and aldehyde; (4) Tropolone acetate is an excellent substrate of the enzyme; (5) Several acetanilide derivatives are susceptible to acetylcholinesterase hydrolysis, although at a considerably slower rate than analogous esters. 2. Materials and methods Acetylcholinesterase from electric eel and acetyl- choline chloride were purchased from Sigma. Pheno- thlazine-10-carbonyl chloride (PTCC, (a) in fig.l) was the product of Aldrich. Commercially available ace- tate esters were of high purity grade. Other acetates were prepared according to reported procedures. For example, 1-butenylacetate was prepared from

Gramicidin S and dodecylamine induce leakage and fusion of membranes at micromolar concentrations.

The effect of the antibiotic gramicidin S and the synthetic cationic amphipath dodecylamine on membranes was studied with large unilamellar vesicles containing phosphatidylcholine and varying concentrations of cardiolipin. Fusion of vesicles composed of equal amounts of the two phospholipids occurred with both drugs at concentrations lower than 10 microM. Fusion was accompanied by leakage of the contents, while higher drug concentrations caused complete loss of vesicle contents. Drug concentrations at least one order of magnitude lower were needed to induce leakage from vesicles containing only phosphatidylcholine. Under these conditions, contents leakage occurred with no measurable aggregation or membrane intermixing. On the other hand, much higher concentrations of both drugs were required to induce leakage from vesicles containing predominantly cardiolipin. Release of contents occurred upon aggregation of the vesicles and collapse of the vesicular organization, as well as formation of paracrystalline structure when dodecylamine was employed or amorphous material when gramicidin A was used. In contradistinction to other model systems, phosphatidylcholine was needed for fusion induced by the cationic amphipaths, and its presence reduced the threshold concentration of the drugs needed to induce leakage of the contents. The similar effects of the two drugs on membranes imply that, at least in these model membranes, the relevant feature of both drugs is only their amphiphatic nature.

The interaction of alkynyl carboxylates with serine enzymes a potent new class of serine enzyme inhibitors

The recently reported alkynyl esters, propynyl benzoate and propynyl p‐methoxybenzoate, were found to interact with a variety of serine enzymes. α‐Chymotrypsin was inhibited very rapidly by an equivalent amount of the esters. Trypsin, elastase and pronase were also inhibited by the esters. On the other hand, liver esterase started to hydrolyse the alkynyl esters rapidly, but the enzyme became inhibited during the course of reaction. The inhibited enzymes exhibited slow reactivation which could be considerably enhanced by hydroxylamine.

The interaction of amiloride with acetylcholinesterase and butyrylcholinesterase.

The diuretic drug amiloride was found to be a powerful inhibitor of the reaction of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) with their specific choline ester substrates. The inhibition constant is in the micromolar range. On the other hand, when added to a mixture of cholinesterase (AChE and BChE) and neutral substrates, amiloride, in some cases, enhanced the reaction rate. The rate of the reaction of butyrylcholinesterase with p‐nitrophenyl butyrate was increased up to 12 fold by amiloride.

The reaction of tertiary amino alcohols with active esters: I. Acylation and deacylation steps

Abstract The reactions of triethanolamine and four other tertiary amino alcohols with six active ester substrates were studied in the pH range 6–10 at 30°C. The reaction products were in all cases the respective O -acyl-amino alcohols. Analysis of the effects of substituents in the leaving group as well as in the acyl moiety of the substrates showed that the ester product was formed by direct attack of the nucleophilic hydroxyl group. Comparison with reactions of tertiary amines with the same substrates supports this conclusion. The reactions of tertiary amino alcohols were also compared with those of zwitterionic quaternary amino alcohols and 3-quinuclidinol, a “rigid” tertiary amino alcohol. On the basis of these comparisons, it is proposed that one of the pathways for the predominant effect of the neutral species of tertiary amino alcohols involves intramolecular general base assistance by the tertiary amino group to the nucleophilic attack of the hydroxylic oxygen on the substrate. The contribution of this pathway to the rate of reaction is evaluated. In several systems the first product of the reaction, an O -acyl-amino alcohol, undergoes relatively rapid deacylation, the overall reaction being thus hydrolysis of active esters, catalyzed by the amino alcohol via an acylation-deacylation mechanism.

On the specificity of porcine elastase

The specificity of porcine elastase (EC 3.4.4.7) has been studied. Ethyl esters derived from benzoyl amino acids with straight side chains are better substrates than those with branched side chains; the best substrate is norvaline ester. In the series of benzoylalanine alkyl esters the alcohol moiety markedly affects the susceptibility. The benzyl ester was found to be the best nonactivated substrate derived from monomeric amino acid. With elastase acylation is rate limiting, in contrast to chymotrypsin and trypsin where deacylation is generally the rate determining step with specific ester substrates.

Alkynyl phosphates are potent inhibitors of serine enzyme

Propynyl, hexynyl and t‐butylethynyl diethyl phosphates were found to be very powerful covalent inhibitors of serine enzymes. Esterases were inhibited with second‐order rate constants of 107–108 M−1 min−1. Most proteases were inhibited with a rate constant of 104–105 M−1 min−1. By inhibiting chymotrypsin with (3‐14C)‐1‐propynyl diethyl phosphate, it was established that inhibition was caused by binding of the phosphate group to the enzyme active site.

The reaction of amino alcohols with active esters: II. Catalysis of the acylation step by metal ions

Abstract The reaction of triethanolamine (TEA) with active substrates— p -nitrophenyl esters and cinnamoyl imidazole (CI)—is catalyzed by divalent heavy metal ions. With Hg 2+ , rate enhancements of 100–1000 (depending on the substrate) were observed, the overall rate constants of substrate decomposition thus exceeding those of spontaneous hydrolysis up to 100,000-fold. The predominant active species at low L:M ratio was found to be the Hg-(TEA) 2 complex. The dependence of the reaction rate upon excess of amino alcohol—at constant Hg 2+ concentration—is attributable to formation of another active complex—Hg-(TEA) 3 . The high reactivity of the system is due to the alcoholate group of metal-bound TEA, whose p K has been lowered by the proximity of the metal ion. This labile nucleophilic alcoholate attacks the substrate causing its alcoholysis and forming O -acyl-TEA. The lability of the metal-alcoholate bond can be enhanced by low concentrations of halide ions, thus causing up to 5-fold additional increase in alcoholysis rate. Higher halide ion concentrations cause inhibition, probably due to formation of inactive HgX 2 molecules. Presumably an important role of the metal ion in metalloenzymes is to affect the decrease in the p K value of a reactive group so that it can exhibit activity under physiological conditions.