pH studies to elucidate the chemical mechanism of penicillin acylase from Kluyvera citrophila.

The variation with pH of the kinetic parameters of penicillin acylase from Kluyvera citrophila has been used to gain information about the chemical mechanism of the reaction catalysed by the enzyme. The pH-dependence of log (V/Km) for penicillin G showed that a group with a pK value over 4.7 must be deprotonated and that a group with a pK value over 9.

Inactivation of penicillin acylase from Kluyvera citrophila by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline: a case of time-dependent non-covalent enzyme inhibition.

Penicillin acylase (PA) from Kluyvera citrophila was inhibited by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), a specific carboxy-group-reactive reagent. Enzyme activity progressively decreased to a residual value depending on EEDQ concentration. Neither enzymic nor non-enzymic decomposition of EEDQ is concomitant with PA inactivation.

Chemical modification of serine at the active site of penicillin acylase from Kluyvera citrophila.

The site of reaction of penicillin acylase from Kluyvera citrophila with the potent inhibitor phenylmethanesulphonyl fluoride was investigated by incubating the inactivated enzyme with thioacetic acid to convert the side chain of the putative active-site serine residue to that of cysteine. The protein product contained one thiol group, which was reactive towards 2,2'-dipyridyl disulphide and iodoacetic acid. Carboxymethylcysteine was identified as the N-terminal residue of the beta-subunit of the carboxy[3H]methylthiol-protein.

Chemical mechanism of lysophosphatidylcholine: lysophosphatidylcholine acyltransferase from rabbit lung. pH-dependence of kinetic parameters.

Lysophosphatidylcholine: lysophosphatidylcholine acyltransferase is an enzyme that catalyses two reactions: hydrolysis of lysophosphatidylcholine and transacylation between two molecules of lysophosphatidylcholine to give disaturated phosphatidylcholine. Following the kinetic model previously proposed for this enzyme [Martín, Pérez-Gil, Acebal & Arche (1990) Biochem. J.

Penicillin acylase mutants with altered site-directed activity from Kluyvera citrophila.

Oligonucleotide-directed mutagenesis has been used to obtain specific changes in the penicillin acylase gene from Kluyvera citrophila. Wild-type and mutant proteins were purified and the kinetic constants for different substrates were determined. Mutations in Met168 highly decreased the specificity constant of the enzyme for penicillin G, penicillin V and phenylacetyl-4-aminobenzoic acid and the catalytic constant kcat for phenylacetyl-4-aminobenzoic acid.

Effect of albumin on acyl-CoA: lysolecithin acyltransferase, lysolecithin: lysolecithin acyltransferase and acyl-CoA hydrolase from rabbit lung.

Acyl-CoA: lysolecithin and lysolecithin: lysolecithin acyltransferases, as well as acyl-CoA hydrolase are important enzymes in lung lipid metabolism. They use amphiphylic lipids as substrates and differ in subcellular localization. In this sense, lipid-protein interactions can be an essential factor in their activity.

Theoretical approach to the steady-state kinetics of a bi-substrate acyl-transfer enzyme reaction that follows a hydrolysable-acyl-enzyme-based mechanism. Application to the study of lysophosphatidylcholine:lysophosphatidylcholine acyltransferase from rabbit lung.

A kinetic model is proposed for catalysis by an enzyme that has several special characteristics: (i) it catalyses an acyl-transfer bi-substrate reaction between two identical molecules of substrate, (ii) the substrate is an amphiphilic molecule that can be present in two physical forms, namely monomers and micelles, and (iii) the reaction progresses through an acyl-enzyme-based mechanism and the covalent intermediate can react also with water to yield a secondary hydrolytic reaction. The theoretical kinetic equations for both reactions were deduced according to steady-state assumptions and the theoretical plots were predicted. The experimental kinetics of lysophosphatidylcholine:lysophosphatidylcholine acyltransferase from rabbit lung fitted the proposed equations with great accuracy.

Essential residues in lysolecithin:lysolecithin acyltransferase from rabbit lung: assessment by chemical modification.

The inhibition of lysolecithin:lysolecithin acyltransferase by several specific reagents was studied. Diisopropyl fluorophosphate (DFP) completely inhibited both activities at a concentration of 4 mM. Activity was not protected by substrate and the enzyme showed a change in circular dichroism spectrum upon treatment with inhibitor.

Thermodynamic profiles of penicillin G hydrolysis catalyzed by wild-type and Met----Ala168 mutant penicillin acylases from Kluyvera citrophila.

The Met-168 residue in penicillin acylase from Kluyvera citrophila was changed to Ala by oligonucleotide site-directed mutagenesis. The Ala-168 mutant exhibited different substrate specificity than wild-type and enhanced thermal stability. The thermodynamic profiles for penicillin G hydrolysis catalyzed by both enzymes were obtained from the temperature dependence of the steady-state kinetic parameters Km and kcat.

Evidence of a pH-dependent conformational change at the active site of lysolecithin:lysolecithin acyltransferase from rabbit lung.

It has been shown that both activities, hydrolysis and transacylation, of lysolecithin:lysolecithin acyltransferase, as well as the conformation of the polypeptide are critically dependent on a pK around 5.8, but the question remains if the same residue(s) is responsible for the conformational change and the loss of activity. In this paper, ultrasonic cavitation is used to study the pH-dependent inactivation.

Essential histidine residues in lysolecithin:lysolecithin acetyltransferase from rabbit lung.

Both activities of rabbit lung lysolecithin:lysolecithin acyltransferase (EC 3.1.1.

The kinetic mechanism of acyl-CoA:lysolecithin acyltransferase from rabbit lung.

Acyl-CoA:lysolecithin acyltransferase is a key enzyme in the deacylation-reacylation pathway of biosynthesis of molecular species of lecithin. However, the mechanism of the reaction has been little studied. In this paper, the kinetic mechanism of acyl-CoA:lysolecithin acyltransferase, partially purified from rabbit lung, is studied.

Substrate selectivity of acyl-CoA:lysolecithin acyltransferase from rabbit lung.

The influence of both polar head and acyl chain of lysophospholipid on the activity of partially purified acyl-CoA:lysolecithin acyltransferase from rabbit lung was studied. It was concluded that the presence of methyl groups on the nitrogen of the base was essential for recognition of lysophospholipid as substrate by the enzyme. With respect to the acyl chain length and saturation, the activity followed the order: 16:0 approximately equal to 18:1 greater than 14:0 greater than greater than greater than 18:0 approximately equal to 12:0.

Mechanism of the reaction catalyzed by acyl-CoA: lysolecithin acyltransferase from rabbit lung. pH studies and chemical modification.

This paper deals with the first attempt to elucidate the chemical mechanism of acyl-CoA: lysolecithin acyltransferase from rabbit lung, a key enzyme in the metabolism of lung surfactant. For this purpose, the pH dependence of kinetic constants as well as the chemical modification of the protein have been studied on a partially-purified preparation. From these experiments, the pKs on which the activity of the enzyme relies have been calculated, giving values of pK1 congruent to 5.

Effect of lipids on activity and conformation of lysolecithin:lysolecithin acyltransferase from rabbit lung.

Lysolecithin:lysolecithin catalyzing two types of reaction, transacylation or hydrolysis, with the same substrate. Both activities have shown to be dependent on several environmental conditions and among them, the presence of lipids. The addition of several classes of lipids activated in all the cases the enzyme, decreasing the hydrolysis/transacylation molar ratio.

Lysolecithin:lysolecithin acyltransferase from rabbit lung. A conformational study.

The enzyme lysolecithin:lysolecithin acyltransferase from rabbit lung has been found to have a relatively disordered conformation in solutions of high ionic strength. The protein exhibited an ordering of structure when salt was suppressed. This conformational change was concomitant with the loss of transacylase activity, the hydrolytic reaction remaining unchanged.

Influence of temperature on stability and activity of lysolecithin acyltransferase and acyl-CoA hydrolase from rabbit lung.

The effect of temperature on the activities of acyl-CoA:lysolecithin acyltransferase and acyl-CoA hydrolase has been studied in microsomal preparations. The enzymes had different thermal stabilities, the hydrolase being more stable. The temperature dependence of enzyme activities was studied either in 4 M NaCl-washed microsomes or NaCl-washed detergent-treated microsomes.

Substrate selectivity of lysophosphatidylcholine: lysophosphatidylcholine acyltransferase from rabbit lung.

The influence of both polar group and acyl chain of lysophospholipids on the lysophosphatidylcholine: lysophosphatidylcholine acyltransferase from rabbit lung was studied. Both, transacylase and hydrolase activities of this enzyme, utilize selectively 1-[1-14C]palmitoyl-sn-glycero-3-phosphocholine when compared with 1-[9,10-3H2]palmitoyl-sn-glycero-3-phosphoethanolamine. Transacylase activity is more selective for lysophosphatidylcholine as acyl acceptor than as acyl donor.

Labelled lipids distribution following [3H]glycerol injection to pregnant rabbits.

1. The time dependent variation in specific activity of serum triglycerides and phospholipids has been studied following an intravenous pulse-labelling with 2-[3H]glycerol to pregnant rabbits. 2.