Aminopeptidase PC from the Hepatopancreas of the Kamchatka Crab Paralithodes camtshatica

G. N. Rudenskaya^1*, A. M. Shmoilov¹, V. A. Isaev¹, A. V. Ksenofontov¹, and S. V. Shvets²

¹School of Chemistry, Lomonosov Moscow State University, Moscow, 119899 Russia, fax: (095) 939-3181; E-mail: Rudenskaya@biorg.msu.su

²Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, Moscow, 117437 Russia; fax: (095) 330-7410

^* To whom correspondence should be addressed.

Received March 1, 1999; Revision received June 30, 1999

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Homogeneous aminopeptidase PC was isolated with yield 67% and purification degree 237 from the hepatopancreas of the Kamchatka crab Paralithodes camtshatica by ion-exchange chromatography on DEAE-Sepharose, hydrophobic chromatography on Phenyl-Sepharose, and gel-filtration on Sephadex G-150. The enzyme is a homodimer with a molecular mass 220 kD (110 × 2). Aminopeptidase PC has pI = 4.1. It hydrolyzes Leu-pNA optimally at pH 6.0 and at the optimum temperature 36-40°C; in the presence of Ca²⁺ the enzyme is stable at pH 5.5-8.0. Aminopeptidase PC is activated by Ca²⁺, Mg²⁺, and Fe²⁺; it is completely inhibited by EDTA, o-phenanthroline, and bestatin. The enzyme contains four Zn atoms per molecule and is therefore a metalloaminopeptidase. The aminopeptidase PC can effectively cleave N-terminal Arg and Lys residues as well as Leu, Phe, and Met residues. K_m and k_cat values for hydrolysis of Leu-pNA were 0.075 mM and 0.19 sec^-1 and for hydrolysis of Arg-pNA 0.078 mM and 0.48 sec^-1, respectively. D-Amino acid residues cannot be cleaved. Thus, aminopeptidase PC of the Kamchatka crab has a mixed substrate specificity which is characteristic of some microbe aminopeptidases. Its N-terminal sequence ESVEIELPEGLSPLV is 46% coincident with that of yeast vacuolar aminopeptidase YSCA.

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KEY WORDS: aminopeptidase PC, metalloaminopeptidase, mixed substrate specificity, isolation

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