Characterization of the metal-binding site in aminopeptidase B.
A recombinant rat aminopeptidase-B (Ap-B) was expressed as a glutathione S-transferase (GST) fusion protein in Escherichia coli BL21 harboring a plasmid pGEX-Ap-B and was purified by glutathione-Sepharose 4B and Q-Sepharose columns. The metal-substituted derivatives of Ap-B, Co(II)- and Cu(II)-Ap-B contain almost 1 mole of cobalt(II) and copper(II) ions per enzyme molecule, respectively. The specific activity of Co(II)-Ap-B is very similar to that of recombinant Ap-B but that of Cu(II)-Ap-B is very low. The dissociation constants of the zinc ions of recombinant Ap-B and of the cobalt ions of Co(II)-Ap-B calculated from the relationships between the free metal ions and the residual enzyme activities are 3.7(+/-1.0)x10(-13) and 4.7(+/-1.0)x10(-12) M, respectively. The EPR parameters (gperpendicular), g// and A//) of Cu(II)-Ap-B were 2.06, 2.27, and 156x10(-4) cm-1. The A// value and the g// of Cu(II)-Ap-B are very similar to those of Cu(II)-thermolysin or Cu(II)-dipeptidyl peptidase III, in which the coordination geometry is a distorted tetrahedral.
Hirose
Junzo
J
Department of Applied Biological Science, Faculty of Life Science and Biotechnology, Fukuyama University, Japan. hirose@fubac.fukuyma-u.ac.jp
Ohsaki
Takamichi
T
Nishimoto
Naoyo
N
Matuoka
Shouji
S
Hiromoto
Takashi
T
Yoshida
Takahide
T
Minoura
Takatosi
T
Iwamoto
Hiroyuki
H
Fukasawa
Kayoko M
KM
D000626:Aminopeptidases / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D008670:Metals / Q000378:metabolism*; D051381:Rats
D008670:Metals; D000626:Aminopeptidases; C019721:aminopeptidase B