P. Estrada

Lysolecithin:Lysolecithin acyltransferase from rabbit lung: Enzymatic properties and kinetic study

Abstract The enzyme lysolecithin:lysolecithin acyltransferase has been isolated from the soluble fraction of rabbit lung and it has been found responsible for two different reactions: (a) transacylation between two molecules of lysolecithin to give dipalmitoylphospha-tidylcholine and glycerophosphorylcholine and (b) hydrolysis of the substrate yielding free fatty acid and glycerophosphorylcholine. Both activities copurify 80-fold with a hydrolysis/transacylation ratio about 2 in all purification steps. The enzyme shows a strong tendency to aggregate with other proteins without loss of activity. The hydrolysis/transacylation ratio is strongly dependent on experimental conditions and these must be carefully controlled if reproducibility is desired. The dependence of this ratio on several parameters is discussed. Based on identical behavior against temperature, β-mercaptoethanol, and iodoacetate and on the appearance of a unique band on electrophoresis, it can be concluded that only one protein is responsible for both activities. The molecular weight ranges between 58,000 and 60,000 D and the amino acid composition shows its acidic character, as described for the enzyme from other sources. The kinetic pattern of both reactions is different and depends on the physical state of the substrate; when the enzyme binds to monomers, the substrate is hydrolyzed to free fatty acid, whereas the binding to micelles favors the transacylation giving phosphatidylcholine. The kinetic constants have been evaluated and a model is proposed for both reactions. The activation energy has been measured and the proximity of the values for the two reactions suggests that the limiting step is the same for hydrolysis and transacylation. The implication of this enzyme in surfactant synthesis is discussed.

Kinetic mechanism of β-glucosidase from Trichoderma reesei QM 9414.

Abstract β-Glucosidase is a key enzyme in the hydrolysis of cellulose to d -glucose. β-Glucosidase was purified from cultures of Trichoderma reesei QM 9414 grown on wheat straw as carbon source. The enzyme hydrolyzed cellobiose and aryl β-glucosides. The doulbe-reciprocal plots of initial velocity vs. substrate concentration showed substrate inhibition with cellobiose and salicin. However, when p-nitrophenyl β- d -glucopyranoside was the substrate no inhibition was observed. The corresponding kinetic parameters were: K = 1.09 ± 0.2 mM and V = 2.09 ± 0.52 μmol · min−1 · mg−1 for salicin; K = 1.22 ± 0.3 mM and V = 1.14 ± 0.21 μmol · min−1 · mg−1 for cellobiose; K = 0.19 ± 0.02 mM and V = 29.67 ± 3.25 μmol · in−1 · mg−1 fro p-nitrophenyl β- d -glucopyranoside. Studies of inhibition by products and by alternative product supported an Ordered Uni Bi mechanism for the reaction catalyzed by β-glucosidase on p-nitrophenyl β- d -glucopyranoside as substrate. Alternative substrates as salicin and cellobiose, a substrate analog such as maltose and a product analog such as fructose were competitive inhibitors in the p-nitrophenyl β- d -glucopyranoside hydrolysis.

Micellar catalysis of polyphenol oxidase in AOT/cyclohexane

Abstract The catalytic behaviour of mushroom polyphenol oxidase has been studied in dioctylsulphosuccinate (AOT)/cyclohexane reverse micelles. The steady-state conditions were accomplished up to 20 min and 17 μg protein in the assay towards 4-methylcatechol and no loss of specific activity was observed relative to aqueous medium. The pH activity profile of the enzyme was kept in reverse micelles as in water, showing a plateau between 5 and 6.5. The stability of polyphenol oxidase to pH was also studied and about 20% inactivation was found in reverse micelles relative to aqueous medium at neutral pHs. Moreover there was a decrease of stability at acidic pHs. The optimum W o obtained was 20 and the enzyme was nearly independent of the surfactant concentration at constant W o . Kinetic studies of polyphenol oxidase towards several substrates showed that the substrate inhibition by p -cresol and 4-methylcatechol observed in buffer was not kept in AOT/cyclohexane reverse micelles. Moreover, the K m increased and the catalytic efficiency ( V / K m ) of the enzyme decreased as the hydrophobicity of substrates was increased.

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. The double-reciprocal plots of initial velocity vs substrate concentration gave two sets of parallel lines which fitted to a ping-pong equation with the following parameters: Km (palmitoyl-CoA) = 8.5 +/- 2 microM, Km (lysolecithin) = 61 +/- 16 microM, and V = 18 +/- 4 nmol/min/mg protein. Inhibition studies by substrates, alternate substrates, and products supported the ping-pong mechanism, although some nonclassical behavior was observed. Palmitoyl-CoA did not inhibit even at concentrations of 100 Km. In contrast, lysolecithin was a dead-end inhibitor with a dissociation constant of Ki = 930 +/- 40 microM. Alternate substrates and CoA showed alternate pathways for the reaction due to the formation of ternary complexes. Dipalmitoylphosphatidylcholine inhibition pointed to an isomerization of the free enzyme prior to the start of the reaction. From these results, an iso-ping-pong kinetic mechanism for lysolecithin acyltransferase is proposed. The kinetic steps of the reaction are correlated with previous chemical studies of the enzyme.

Chemical mechanism of β-xylosidase from Trichoderma reesei QM 9414: pH-dependence of kinetic parameters

The variation of kinetic parameters of beta-xylosidase from Trichoderma reesei QM 9414 with pH was used to elucidate the chemical mechanism of the p-nitrophenyl beta-D-xylopyranoside hydrolysis. The pH-dependence of V and V/K(m) showed that a group on the enzyme with a pK value of 3.20 must be unprotonated and a group with a pK value of 5.20 must be protonated for activity and both are involved in catalysis. Solvent-perturbation studies indicated that these groups are neutral acid type. Temperature dependence of kinetic parameters suggested the stickiness of the substrate at lower temperatures than the optimum and the calculated ionization enthalpies pointed to carboxyl groups as responsible for both pKs. Chemical modification with triethyloxonium tetrafluoroborate and protection with the substrate studies demonstrated essential carboxyl groups on the enzyme. Profiles of pK(i) for D-gluconic acid lactone indicated that a group with a pK value of 3.45 must be protonated for binding and it has been assigned to the carboxyl group of D-gluconic acid formed by lactone ring breakdown in solution.

Immobilization of β-xylosidase from Trichoderma reesei QM 9414 on nylon powder

β-xylosidase was isolated and partially purified from Trichoderma reesei QM 9414 grown on wheat straw as the sole carbon source. The enzyme was attached covalently to nylon powder and the optimal conditions for the immobilization procedure were determined. The optimum pH value (4.0) was conserved but the optimal temperature for catalysis (55°C) decreased 5°C after immobilization; the activation energy and thermal stability also decreased. Kinetics towards xylobiose and synthetic substrates in batch demonstrated that no saturation was reached using the immobilized derivative with the optimal protein retention, whereas saturation with p-nitrophenyl β-xylopyranoside (pNPX) was reached in a reactor loaded with a low protein retention derivative, giving KM = 1 mM pNPX and Vmax = 0.35 μmol/min/mg protein. We investigated the operating conditions under which the reactor must work to reach the highest specific activity and this corresponds to 37.6% substrate conversion. A theoretical calculus will allow us to des...

Polyphenol oxidase in reverse micelles of AOT/cyclohexane : A thermostability study

Polyphenol oxidase catalysing the oxidation of 4-methylcatechol in reverse micelles of AOT/cyclohexane had an optimum temperature 15 °C higher than in aqueous medium. However, the enzyme lost stability when it was preincubated in reverse micelles in the absence of substrate regardless of the temperature although the effect was more pronounced at higher temperatures. The thermostability of polyphenol oxidase is higher when it is injected in reverse micelles containing buffer than when injected in initially empty micelles. Moreover, the thermostability of polyphenol oxidase in reverse micelles is strongly dependent on the size of the micelles, the bigger the micelle the greater the stability. The thermoinactivation of the enzyme follows a monomolecular process characteristic of a conformational change so the protein is protected by ligands towards inactivation. p-Nitrophenol as competitive inhibitor and acetyl tyrosine ethyl ester as alternate substrate increase the half-life of the polyphenol oxidase by about 2.5 and 4 times respectively. This finding may allow the use of the enzyme at higher temperatures with a gain in its stability. © 2001 Society of Chemical Industry

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. We have studied the effect of albumin, as lipid-binding protein model, in the activities of these enzymes. Acyl-CoA hydrolase was inhibited in the presence of albumin, whereas acyl-CoA : lysolecithin acyltransferase showed a complex effect of activation depending on both albumin concentration and palmitoyl-CoA/lysolecithin molar ratio. Lysolecithin : lysolecithin acyltransferase was affected differentially on its two activities. Hydrolysis remained unaffected and transacylation was inhibited by albumin. These results are consequence of the interaction of albumin with both lipidic substrates that changes their critical micellar concentration.

Self-aggregation of a recombinant form of the propeptide NH2-terminal of the precursor of pulmonary surfactant protein SP-B: a conformational study

A recombinant form of the peptide N-terminally positioned from proSP-B (SP-BN) has been produced in Escherichia coli as fusion with the Maltose Binding Protein, separated from it by Factor Xa cleavage and purified thereafter. This protein module is thought to control assembly of mature SP-B, a protein essential for respiration, in pulmonary surfactant as it progress through the progressively acidified secretory pathway of pneumocytes. Self-aggregation studies of the recombinant propeptide have been carried out as the pH of the medium evolved from neutral to moderately acid, again to neutral and finally basic. The profile of aggregation versus subsequent changes in pH showed differences depending on the ionic strength of the medium, low or moderate, and the presence of additives such as L-arginine (a known aggregation suppressor) and Ficoll 70 (a macromolecular crowder). Circular dichroism studies of SP-BN samples along the aggregation process showed a decrease in α-helical content and a concomitant increase in β-sheet. Intrinsic fluorescence emission of SP-BN was dominated by the emission of Trp residues in neutral medium, being its emission maximum shifted to red at low pH, suggesting that the protein undergoes a pH-dependent conformational change that increases the exposure of their Trp to the environment. A marked increase in the fluorescence emission of the extrinsic probe bis-ANS indicated the exposure of hydrophobic regions of SP-BN at pH 5. The fluorescence of bis-ANS decreased slightly at low ionic strength, but to a great extent at moderate ionic strength when the pH was reversed to neutrality, suggesting that self-aggregation properties of the SP-BN module could be tightly modulated by the conditions of pH and the ionic environment encountered by pulmonary surfactant during assembly and secretion.

Kinetic mechanism of β-xylosidase from Trichoderma reesei QM 9414

Abstract β-Xylosidase is a key enzyme in the hydrolysis of xylobiose to d -xylose. The enzyme was purified from cultures of Trichoderma reesei QM 9414 grown on wheat straw as a carbon source. β-Xylosidase shows the highest catalytic efficiency towards xylobiose and the highest affinity for 4-methylumbellyferyl-β- d -xylopyranoside, an arylxyloside whose aglycone is very hydrophobic. Studies of inhibition by products on the p -nitrophenyl-β- d -xylopyranoside kinetics and by an alternative product, 4-methylumbelliferone, supports an Ordered Uni Bi mechanism for the reaction catalysed by β-xylosidase. Competitive inhibition by a product analog, D-glucono-1,5-lactone is also compatible with the proposed mechanism.