Aminopeptidase PC from the hepatopancreas of the Kamchatka crab Paralithodes camtshatica.
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 x 2). Aminopeptidase PC has pI = 4.1. It hydrolyzes Leu-pNA optimally at pH 6.0 and at the optimum temperature 36-40 degrees 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. Km and kcat 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.
Rudenskaya
G N
GN
School of Chemistry, Lomonosov Moscow State University, Moscow, 119899, Russia. Rudenskaya@biorg.msu.su.
Shmoilov
A M
AM
Isaev
V A
VA
Ksenofontov
A V
AV
Shvets
S V
SV
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000626:Aminopeptidases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D000818:Animals; D003386:Brachyura / Q000201:enzymology* / Q000235:genetics; D002847:Chromatography, Agarose; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004064:Digestive System / Q000201:enzymology; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D000596:Amino Acids; D008670:Metals; D000626:Aminopeptidases