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-methylfoumaryl-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 derivatives employed. From the kinetic constants with four unblocked substrates, carp cathepsin H had the highest affinity toward Leu-MCA with a K-m value of 35.4 mu M. 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 V-max and K-cat 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. Copyright (C) 1997 Elsevier Science Inc.