Oligomeric Structure and Substrate Induced Inhibition of Human Cathepsin C

Abstract

Cathepsin C has been purified from human kidney by a modified procedure. Human cathepsin C was isolated as pure protein with a pI close to 6.0. The enzyme was shown to have a molecular mass of 200 kDa and to consist of four identical subunits, each composed of three different polypeptide chains, two of them disulfidebound. Their NH 2 -terminal amino acid sequences were determined. Two chains showed pronounced similarity with the heavy and light chains of other papain-like cysteine proteinases, whereas the third one corresponded to the prosequence of the enzyme, thus showing that a substantial part of the proregion remains bound in the mature enzyme. The kinetics of substrate hydrolysis deviated substantially from standard Michaelis-Menten kinetics, demonstrating substrate inhibition at higher substrate concentrations. These data are explained by a sequential cooperative interaction model, where an enzyme molecule can bind up to four substrate molecules but where only the binary enzymesubstrate complex is catalytically active. Substrate inhibition was observed over the whole range of pH activity. From the pH activity profile it can be concluded that at least three ionizable groups with pK a values 4.2, 6.8, and 7.7 are involved in substrate hydrolysis. Human cathepsin C thus appears to differ qualitatively from other cysteine proteinases of different origin.

Interaction of human cathepsin C with chicken cystatin

“…1) which was identical with that obtained for the enzyme prepared by using the earlier method [4], indicating that the isolated enzyme was pure. Cathepsin C is an oligomeric enzyme, each subunit consisting from three different polypeptide chains [4]. From 500 g of spleen we obtained 8 mg of cathepsin C. This is a much higher yield than those obtained by other methods, which included several chromatography steps [4,25,26].…”

“…A product gave a pattern on SDS-PAGE ( Fig. 1) which was identical with that obtained for the enzyme prepared by using the earlier method [4], indicating that the isolated enzyme was pure. Cathepsin C is an oligomeric enzyme, each subunit consisting from three different polypeptide chains [4].…”

“…Like other cysteine proteinases cathepsin C is involved in intracellular protein degradation [2] and is found in a variety of human and rat tissues [3]. Cathepsin C is an oligomeric enzyme, consisting of four identical subunits, each consisting of a heavy and a light chain and a large proregion part, thus differing from other papain-like cysteine proteinases [4]. It is active in the pH range between 3.5 and 8.0 [5] and cleaves peptides and proteins with an unsubstituted amino terminus [6][7][8][9][10][11], as well as certain synthetic substrates with a blocked amino terminus [12].…”

Molecular Cloning, Chromosomal Localization, and Expression of Murine Dipeptidyl Peptidase I

“…Asterisks indicate the conserved cysteine, histidine, and asparagine residues in the active site. Underlined sequences indicate the NH 2 -terminal sequences of the pro, mature, and light chains obtained by partial protein sequencing of DPPI (isolated from human spleen) (28). Arrowheads indicate the possible amino termini of the proregion, heavy, and light chains.…”

“…There are differing reports concerning the subunit composition of the enzyme, ranging from dimers to hexamers. Most studies suggest that the monomeric subunit is composed of a heavy chain and light chain (both originating from the mature region of the protein); however, a recent report presented evidence that active human DPPI consists of four identical subunits, each composed of three different polypeptide chains, two of them disulfide-linked (28). Two of the chains showed pronounced similarity to the heavy and light chains of the other papain-like cysteine proteases, whereas the third chain corresponds to the proregion of the enzyme.…”