Substrate specificity of carp Cyprinus carpio cathepsin H with methylcoumarylamide substrates.
Cathepsin H was purified from the crude extract of carp (Cyprinus carpio) hepatopancreas by a reformed method involving six stages, and the specific activity increased about 11,500-fold with a 23% recovery. Of varying fluorescent synthetic substrates tested, carp cathepsin H possessed an ability to hydrolyze four N-terminal unblocked substrates those are composed of a single amino acid bound to 4-methylcoumaryl-7-amide (MCA), namely Leu-MCA, Arg-MCA, Lys-MCA and Ala-MCA. In contrast the enzyme was only marginally able to hydrolyze an unblocked substrate such as Pro-Phe-Arg-MCA and totally unable to degrade all blocked derivative employed. From the kinetic constants with four unblocked substrates, car cathepsin H had the highest affinity toward Leu-MCA with a Km value of 35.4 microM. Besides both the hydrolytic rates and molecular activities of the enzyme decreased from Lys-MCA > Arg-MCA > Ala-MCA > Leu MCA as judged by their Vmax and Kcat values, respectively. Otherwise, optimal pHs for hydrolysis of cathepsin H were different for four substrates. The enzyme exhibited maximum level of the activity at pH 6.5 for Arg-MCA and Lys-MCA and at pH 7.0 for Leu-MCA and Ala-MCA.
Aranishi
F
F
National Research Institute of Fisheries Science, Yokohama, Japan. aranishi@nrifs.affrc.go.jp
Hara
K
K
Osatomi
K
K
Ishihara
T
T
D000818:Animals; D002347:Carps / Q000378:metabolism*; D056655:Cathepsin H; D002403:Cathepsins / Q000302:isolation & purification / Q000378:metabolism*; D003374:Coumarins / Q000737:chemistry; D003546:Cysteine Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D015394:Molecular Structure; D010179:Pancreas / Q000201:enzymology; D013379:Substrate Specificity
D003374:Coumarins; D002403:Cathepsins; D003546:Cysteine Endopeptidases; D056655:Cathepsin H