Armin Mehlich

Semisynthetic engineering of proteinase inhibitor homologues.

A semisynthetic approach to modulate the inhibitory specificity of aprotinin, the Kunitz trypsin inhibitor from bovine mast cells, is described. By the use of peptide-chemical procedures a single amino acid of its reactive site can be replaced by any other coded or non-coded amino acid. Thus, a series of aprotinin homologues have been prepared which demonstrate the individual contribution of a single side chain to the inhibition of a particular target proteinase and enable specific inhibitors to be designed.

Semisynthesis of Arg15, Glu15, Met15, and Nle15-aprotinin involving enzymatic peptide bond resynthesis

The semisynthesis of homologues of aprotinin, the bovine pancreatic trypsin inhibitor, is described. The P1 lysine15 residue was replaced by two methods. The first procedure, which consisted of two enzymatic steps for the incorporation of other amino acids has previously been described. The second approach consisted of six steps of both enzymatic and chemical nature. The modified inhibitor, in which the lysine15-alanine16 peptide bond is hydrolyzed, was used as the starting material. All carboxyl groups of the modified inhibitor were esterified with methanol; the lysine15 methylester group was then selectively hydrolyzed. Afterward, lysine15 itself was split off. Arginine, glutamic acid, methionine, andl-2-aminohexanoic acid (norleucine, Nle) were incorporated using water-soluble carbodiimide combined with an acylation catalyst. The methylester group was used to prevent polymerization. The reactive-site peptide bonds were resynthesized using either chymotrypsin or trypsin.

Aprotinin derivatives with chromophoric leaving groups can be used as highly selective active-site titrants for serine proteinases and permit the determination of kinetic constants of enzyme-inhibitor complexes.

This paper reports a novel and valuable approach to active-site titration. The starting substance for the preparation of the active-site titrants is aprotinin (bovine pancreatic trypsin inhibitor) in which the reactive-site peptide bond, Lys15-Ala16, is split. Two cystine disulfide bonds hold together the two peptide chains. The Lys15 of the reactive site is substituted by arginine-, phenylalanine- and valine-4-nitroanilide or by valine-7-amido-4-methylcoumarin. The different incorporated amino acid residues correspond to different specificities against serine proteinases. Serine proteinases with suitable specificity are able to remove 4-nitroaniline or 7-amino-4-methylcoumarin from these aprotinin derivatives while at the same time resynthesis of the reactive-site peptide bond occurs. The proteinase is then trapped in a stable enzyme-inhibitor complex, which prevents the proteinase from releasing further leaving groups. The quantity of 4-nitroaniline or 7-amino-4-methylcoumarin, which can be assayed spectrophotometrically or fluorometrically is equimolar to the quantity of proteinase used and trapped. The aprotinin derivatives with an incorporated Phe15 or Val15 residue are highly specific for chymotrypsin or for elastase from human leukocytes, respectively. The kinetic constants kon and koff of the enzyme-inhibitor complexes, and hence the equilibrium dissociation constants, can be calculated from the respective titration curves.

Semisynthetic Inhibitors of Human Leukocyte Elastase and their Protective Effect on Lung Elastin Degradation in Vitro

Proteinases and the control of their activity by inhibitors are involved in many biological processes (1). A proteinase/proteinase inhibitor imbalance hypothesis has been proposed to account for several pathological situations such as rheumatoid arthritis (2,3) or pulmonary emphysema (4). The latter is a reduction of lung function due to a greaterproteolytic degradation of elastin either by an increase of proteinase release, deactivation of local proteinase inhibitors or a hereditary deficiency of α1-proteinase inhibitor (α1-PI). A disturbance of the elastin cross linking also appears to be involved (5,6). The elastase of human polymorphonuclear leukocytes (HLE) (7), which was shown to produce emphysema in several animal models possibly plays a prominent role (8). The appearance of a HLE-induced emphysema could be prevented by administering a corresponding proteinase inhibitor (9). Inhibitors of this elastase and their interaction with the enzyme, especially in the presence of the physiological substrate elastin, are thus of particular interest.