Arieh Berger

On the active site of proteases. III. Mapping the active site of papain; specific peptide inhibitors of papain

Abstract It was found that in papain one of the subsites of the active site - namely “S 2 ” - specifically interacts with phenylalanine residues. The evidence is that: (a) the presence of Phe in positions three or higher from the C-terminal of the substrate enhances the susceptibility of a peptide to hydrolysis; (b) Phe directs the enzymic attack to the bond next-but-one to it towards the C-terminal; (c) peptides containing Phe as the second residue from the C-terminal are strong competitive inhibitors of papain ( K j ∼ 10 3 M −1 ) . It is assumed that these inhibitors occupy part of the active site (S 1 , S 2 , etc.) in the same manner as substrates. Thus they may help, by means of X-ray analysis of their complexes with papain, to show how peptide chains are bound to the active site of the enzyme.

Polymers of tripeptides as collagen models: II. Conformational changes of poly(L-prolyl-glycyl-L-prolyl) in solution

An ordered sequence polymer of the structure H(Pro.Gly.Pro)n OH was synthesized as a collagen model. Fractionation by gel filtration gave samples of average molecular weights between 1000 and 12,000. The polymer showed in aqueous solution ordered (helical) conformation which could be destroyed by heating or by the addition of a number of salts. Extent of helicity and sharpness of thermal transitions increased with increasing molecular weight. On the basis of optical rotation measurements, it was concluded that the observed structure is of the poly- L -proline II type, stabilized by co-operative hydrogen-bonding leading to side-to-side association of chain sections. The resulting structure is probably the triple-stranded helix observed in the solid state with this polymer, but alternatives (anti-parallel arrangement, double-strand structure) could not be excluded. The results obtained show that the replacement of every third proline residue in poly- L -proline by a glycine residue, thus introducing one CONH group per tripeptide unit, is sufficient to stabilize a collagen-like structure in solution as well as in the solid state.

On the size of the active site in proteases II. Carboxypeptidase-A

Abstract The size of the active site of carboxypeptidase-A was investigated by studying the kinetics of hydrolysis of peptides of L-alanine, D-alanine and L-phenylalanine, as well as of a number of their N-benzyloxycarbonyl, N-acetyl, N-phenylproprionyl and N-methyloxycarbonyl derivatives. From a comparison of the various kinetic parameters ( K m, kcat) it was concluded that the active site of this enzyme extends over about 18 A. The binding area can be divided into 5 “subsites”, each accommodating one amino acid residue (or blocking group) of the substrate. By comparing K m values of pairs of substrates containing either a methyl or a benzyl side-chain in equivalent positions, it was shown that the binding area as a whole has a larger affinity towards the aromatic residues. Substitution of a D-residue for an L-residue reduced kcat values rather than K m. In addition a remarkable affinity for the urethane-grouping located specifically at subsite S3 was found. A methyloxycarbonyl or benzyloxy — carbonyl group occupying this subsite caused a 5-fold increase in K m as compared with an acetyl or phenylproprionyl group, respectively. Methyloxycarbonyl or benzyloxycarbonyl groups in subsites S2 or S4 showed no such effect.

The antibacterial action of some basic amino acid copolymers

Abstract 1. 1. Twenty-two poly-α-amino acids were prepared, composed of one or several of the following amino acids: alanine, leucine, valine, phenylalanine, proline, sarcosine, lysine, and ornithine. 2. 2. The copolymers containing ornithine or lysine were found to inhibit the growth of E. coli and M. pyogenes in synthetic and semisynthetic media, respectively, in the range of 2.5–15 μg./ml. 3. 3. The ornithine-leucine-containing copolymers acted as highly bactericidal agents toward E. coli and M. pyogenes and caused rapid release of inorganic phosphorus from both organisms, resembling gramicidin S in this respect. 4. 4. Strains of M. pyogenes resistant toward gramicidin S and some of the ornithine-leucine copolymers were developed. A cross-resistance relationship was established between gramicidin S and leucine—ornithine copolymers. 5. 5. The bacterial spectrum and the toxicity of the most active copolymers were determined. 6. 6. The role of the ornithine and leucine residues in determining the antibacterial properties of the copolymers used is discussed.

Multi-chain polyamino acids containing glutamic acid, aspartic acid and proline

Abstract 1. 1. A number of multi-chain polyamino acids were synthesized by reacting N-carboxyamino acid anhydrides with multi-functional amines such as polylysine or preformed multi-chain polyamino acids. The purpose of this synthesis was to obtain a family of compounds which could serve as protein models in the study of hydrodynamic, electrochemical and conformational properties. 2. 2. It was found that in this method of synthesis linear polymers may be formed as by-products and methods were worked out for the purification of the multi-chain fraction. Molecular weights of the materials obtained ranged from 50 000 to 1 500 000. There was good agreement between molecular weights as determined by physical methods and those calculated from the degree of polymerization of the polylysine “backbone” and total amino acid analysis. 3. 3. Applying the “equivalent ellipsoid” approach to hydrodynamic measurements reasonable values were calculated for the length of the resulting molecules, but it was concluded that a more refined theory is necessary to describe the overall shape of the multi-chain compounds. 4. 4. Electrochemical studies showed that various ionizable groups had dissociation constants similar to those observed for the corresponding groups in proteins. There seems to be evidence that the dissociation constant are influenced more by next-neighbour effects than by the general charge of the molecule. 5. 5. The side chains of the multi-chain polymers are able to assume helical conformation if long enough. Solvent-induced helix-coil transitions were observed with poly-β-benzyl- L -aspartate and poly-γ-benzyl- L -glutamate side chains. Poly- L -proline side chains were shown to be able to assume the poly- L -proline I or poly- L -proline II conformation (according to the solvent employed) if the average chain length was larger than six.

On the size of the active site in proteases: Pronase

Abstract The size of the active site of the enzyme Pronase has been determined by the method of diastereoisomeric replacement and shown to be five amino acid residues in length. The application of this method for measuring the size of the active site to broad spectrum enzymes is demonstrated.

Evaluation of Multichain Polyglutamic Acid as a Possible Plasma Expander

Summary Some biological properties of 3 batches of a multichain polyamino acid, multi-(copoly-L-glutamyl-L-aspartyl) poly-L-lysine, were examined in vivo in dogs and in rabbits. The polymer was administered intravenously as 2 to 3% solutions of its sodium salt by one or by repeated injections. In some animals hypovolemia was produced by arterial bleeding prior to infusion. A low-molecular weight fraction of the polymer, amounting to about 10 to 20%, was excreted in the urine within a few hours. The rest of the injected polymer was not excreted in the urine, and disappeared gradually from the blood, with a constant half-concentration time of about 48 hours. As shown by its influence on blood pressure and on hematocrit, the polymer was able to combat the manifestations of hypovolemic shock. However, severe toxic manifestations appeared in many animals, consisting of diarrhea, vomiting, loss of appetite, a transient drop in blood pressure and a tendency to capillary bleeding. Progressive weakness of the animals was observed, leading to their death. The cause of toxicity of these polypeptides has not been found. Possibly their multiple negative charges may be responsible. It appears worthwhile to examine non-charged or isoelectric polyamino acids with regard to their plasma expanding properties and their toxicity.