On the size of the active site in proteases. I. Papain.

Acetylornithinase of Escherichia coli: partial purification and some properties.

The preparation and properties of two new chromogenic substrates of trypsin.

https://www.farm.ucl.ac.be/Full-texts-FARM/Danhier-2010-2.pdf

To exploit the tumor microenvironment: Passive and active tumor targeting of nanocarriers for anti-cancer drug delivery

The spectrophotometric procedure proposed by Schwert and Takenaka for the assay of chymotrypsin and trypsin has been modified and extended to include the application to N-benzoyl-L-tyrosine ethyl e...

A modified spectrophotometric determination of chymotrypsin, trypsin, and thrombin.

In earlier reports from this laboratory, a series of new synthetic substrates for pepsin was described.'-' These compounds are of the general type Z-His-X-YOAI\\e4 where X and Y are the residues of amino acids such as L-phenylalanine, p-nitro-L-phenylalanine, L-tyrosine, L-tryptophan, and L-leucine; the enzymic action is limited to the cleavage of the X-Y bond. One of the substrate analogues prepared during the course of this work, Z-His-Phe-Pol, was found to be resistant to the-action of pepsin3 and to be a competitive inhibitor of the enzyme.5 The close structural similarity of this inhibitor to the sensitive substrate Z-HisPhe-Phe-OSAe (replacement of the terminal COOCH3 group by a CH20H group), and the fact that the K, for Z-His-Phe-Pol is similar to the Km for Z-His-PhePhe-O\\e,5 suggested that such a derivative of L-phenylalaninol would be useful for the study of its binding at the catalytic site of pepsin. The derivative chosen was Z-His-Phe(NO2)-Pol, because of its high molecular extinction coefficient and because kinetic data have shown that Z-His-Phe-Phe-O\\Ike and Z-His-Phe (NO2)Phe-OA/le are cleaved by pepsin at similar rates.3 To determine the binding of Z-His-Phe(NO2)-Pol to pepsin, the gel-filtration method6 as refined in this laboratory7 was employed. In this method, a Sephadex column is equilibrated with the substance of low molecular weight, and the protein (dissolved in the solution used to equilibrate the column) is applied. Elution of the column with the same solution as that used for equilibration leads to the emergence of the protein at the excluded volume of the column, followed in the elution diagram by a trough whose area gives a measure of the amount of small molecule bound by the protein. The use of Z-His-Phe(NO2)-Pol as the small molecule facilitates the spectrophotometric monitoring of the effluent solution, because the molecular extinction coefficient of this compound is 9600 at 278.5 M/1 (the absorption maximum at pH 4). In the present communication data are presented in favor of the conclusion that pepsin has a primary binding site of high affinity for Z-His-Phe(N02)-Pol, and in addition binds the small molecule more weakly at secondary sites. That this primary binding site is located at the catalytic site of the enzyme is indicated by the finding that treatment of pepsin with the diazo ketone L-1-diazo-4-phenyl-3tosylamidobutanone (DPTB) abolishes the high-affinity binding at the primary site. Earlier studies in this laboratory had shown that this diazo ketone specifically inhibits the catalytic activity of pepsin as a proteinase (substrate, hemoglobin), as a peptidase (substrate, Z-His-Phe(NO2)-Phe-O1\\Ie), and as an esterase (substrate, Z-His-Phe(N02)-Pla-OZAe); complete inactivation is achieved by the reaction of one molecule of the diazo ketone per molecule of pepsin.3 8 The binding of Z-His-Phe(N02)-Pol to pepsin previously treated with acetyl imidazole also has been examined. Such treatment has been shown to cause a marked decrease in the rate of peptic cleavage of hemoglobin, and a marked

Joseph S. Fruton

Papers

The substrate binding site of pepsin.

The activation of intracellular proteinases

Polymerization reactions catalyzed by intracellular proteinases. iv. factors influencing the polymerization of dipeptide amides by cathepsin c.

Gel filtration studies on the binding of peptides to pepsin.

Utilization of methionine derivatives by a mutant strain of Escherichia coli.