Oligomeric structure and substrate induced inhibition of human cathepsin C.
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 disulfide-bound. Their NH2-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 enzyme-substrate 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 pKa 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.
Dolenc
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I
Department of Biochemistry and Molecular Biology, J. Stefan Institute, Ljubljana, Slovenia.
Turk
B
B
Pungercic
G
G
Ritonja
A
A
Turk
V
V
D000595:Amino Acid Sequence; D000818:Animals; D020789:Cathepsin C; D002403:Cathepsins / Q000737:chemistry*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004151:Dipeptides; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D008970:Molecular Weight; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D004151:Dipeptides; D004792:Enzyme Precursors; C095565:glycylphenylalanine 4-methoxy-2-naphthylamide; D002403:Cathepsins; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases; D020789:Cathepsin C; C095231:preprocathepsin C