Hubert Gaertner

Increased activity and stability of poly(ethylene glycol)-modified trypsin.

The reaction of trypsin with activated monomethoxypoly(ethylene glycol) with various molecular masses led to the development of a series of poly(ethylene glycol)-modified trypsins (PEG-trypsins). On determining the catalytic properties of PEG-trypsin using N-benzoyl-L-arginine p-nitroanilide as a substrate, a three- to fourfold increase in the maximal velocity of hydrolysis was found to occur, whatever the size of the PEG moiety used. PEG-trypsin with higher molecular mass moieties showed lower Michaelis constant values. The activation of trypsin was neither reversed by nucleophiles such as hydroxylamine, nor prevented when modification was carried out in the presence of benzamidine or in the presence of the polypeptidic soybean trypsin inhibitor. Chemical modification of about 80% of the free amino groups with PEG chains significantly improved the resistance to heat and detergents. This might result from the formation of a highly hydrogen-bonded structure around the enzyme.

Subtilisin-catalysed peptide synthesis and transesterification in organic solvents

SummarySubtilisin from Bacillus subtilis was modified with polyethylene glycol (PEG), or adsorbed either on celite or porous glass, or directly used as a suspended powder to catalyse peptide synthesis and transesterification reactions in organic solvents. The rather low yield of peptide synthesis probably resulted from the enzyme tendency to catalyse hydrolysis and transesterification side reactions. The kinetics of transesterification catalysed by PEG-subtilisin was consistent with a ping-pong mechanism modified by a hydrolytic branch. Initial rates of transesterification were found to be dependent on alcohol and organic base concentrations in the reaction mixture. The high affinity of benzyloxycarbonyl-l-serine-methyl ester for the enzyme indicated that a change in substrate specificity of subtilisin occurred in organic phase. The 50-fold increase in the rate of synthesis of benzyloxycarbonyl-l-serine-l-phenylalanine amide which was observed when PEG-subtilisin was used instead of immobilized or powdered enzyme, suggested that a higher flexibility of the polypeptide chain modified by the covalent attachment of a number of soluble PEG moieties occurred in organic solvents. This also resulted in a lower stability of PEG-subtilisin at high temperature.

Unusual specificity of polyethylene glycol-modified thermolysin in peptide synthesis catalyzed in organic solvents

SummaryPolyethylene glycol-modified thermolysin was found to efficiently catalyze peptide synthesis in organic solvents. As in aqueous media, the reaction occurred through a rapid equilibrium random bireactant mechanism. However, the substrate specificity of modified thermolysin was actually changed since hydrophilic as well as acidic amino acids were better carboxyl group donors than hydrophobic residues, contrary to what is observed in both the enzyme-catalyzed synthesis and hydrolysis of peptide bonds in water.

Papain-Catalyzed Peptide Synthesis and Oligomerization of Amino Acid Amides in Organic Solvents

Papain, either modified with polyethylene glycol or adsorbed on porous glass beads, was used to catalyze peptide bond formation between N-acyl-L-amino acid esters and L-phenylalanine amide in organic solvents. Optimal reaction parameters of the modified enzyme were derived from the dependence of its activity upon water and mercaptoethanol concentrations in the reaction mixture. Under the same experimental conditions, immobilized papain was found to be considerably less efficient than modified papain in catalyzing the synthesis of N-α-benzoyl-L-lysine-L-phenylalanine amide in 1,1,1,-trichloroethane. Moreover, the pH at which the enzyme has been lyophilized prior to its adsorption on glass beads also had an important influence on the activity. Peptide synthesis yields higher than 90% were obtained with both papain forms when either basic or acidic amino acids were used as acyl-donor residues, while neutral and aromatic residues were rather poor substrates but initiated a polymerization reaction.

Papain-catalyzed polymerization of amino acids in low water organic solvents

SummaryPolyethylene glycol-modified papain catalyzed the oligomerization of amino acid amides in toluene. The water content of the organic solvent was a critical factor determining the extent of polymerization. Under optimized conditions, lysine and phenylalanine oligomers containing up to 10 residues were obtained. In sharp contrast to what is observed in aqueous media, hydrophilic and basic amino acid derivatives resulted in higher reaction yields than hydrophobic amino acid derivatives.

Transport of N-α(or N-ϵ)-L-methionyl-L-lysine and acetylated derivatives across the rabbit intestinal epithelium

Abstract Intestinal absorption constitutes an important step in the biological utilization of L -Methionine and N-Acetyl- L -methionine covalently bound to ϵ-NH2 groups of food protein lysyl residues. The transport of synthesized and purified N-α-(or N-ϵ-) L -Methionyl- L -lysine and N-α-(or N-ϵ-)Acetyl- L -methionyl- L -lysine was studied in vitro in rabbit ileum mounted in the Ussing chamber and compared to that of both free L -Methionine and L -Lysine or both free N-Acetyl- L -methionine and L -Lysine. Addition of all solutes to the mucosal reservoir, except N-ϵ-Acetyl- L -methionyl- L -lysine, led to an increase in the short-circuit current, the lowest response obtained by N-α-Acetyl- L -methionyl- L -lysine. In all cases, only the constitutive amino acids were recovered in the serosal chamber. When free L -Methionine and L -Lysine were added together to the mucosal reservoir, comparable fluxes were obtained: when any of the dipeptides were added, transport of lysine was the highest. Although the mucosal reservoir disappearance of N-α- L -Methionyl- L -lysine was faster than that of N-ϵ- L -Methionyl- L -lysine, the mucosal to serosal fluxes of their constitutive amino acids were not significantly different. However, the transepithelial flux of L -Methionine originating from the dipeptides was 40–50% less than that of the free amino acid, whereas L -Lysine flux was unchanged. Substantial tissue oxidation of L -Methionine unlike L -Lysine, which are slowly released from peptide hydrolysis by the brush border membrane aminopeptidases, likely altered the amino acid transport. Moreover, the observed higher transepithelial flux for L -Lysine relative to L -Methionine when both were derived from the dipeptides may have resulted from intracellular L -Methionine stimulation of L -Lysine absorption. As compared to the normal di- and isodipeptides, lower net fluxes of L -Methionine, and to a lesser extent of L -Lysine, were observed from the acetylated peptides, the former being zero even in the case of N-ϵ-Acetyl- L -methionyl- L -lysine. The results demonstrate the significant contribution of brush border aminopeptidases to the intestinal absorption of N-ϵ- L -Methionyl- L -lysine in the rabbit. Our findings also suggest that the availability of amino acids from acetylated peptides is limited by their transport rates and/ or their hydrolysis from cytosolic N-acylpeptide hydrolase and N-acylase.

Hydrolysis of the isopeptide bond of ϵ-N-L-methionyl—L-lysine by intestinal aminopeptidase N

Amide bonds involving e-amino groups of lysine and/or w-carboxyl groups of aspartic and glutamic acid are generally called ‘isopeptide bonds’ in contrast to the ol-peptide bonds which normally link amino acids residues in proteins and peptides. These bonds are known to be responsible for the covalent crosslinking of several proteins [ I] and other natural products such as the peptidoglycan network of bacteria cell walls [2]. They may also be formed artifactually by degradative reactions within or between proteins during processing [3] and by chemical grafting of essential amino acids through their cu-carboxyl to the e-amino group of lysine residues in food proteins [4,5]. Here, only latter type of isopeptide bond is considered. for pure aminopeptidase N from pig and rabbit intestine. The e-dipeptide was also used to test the properties of the different subsites assumed to be present in the binding site of the enzyme.