Substrate specificities of deuterolysin from Aspergillus oryzae and electron paramagnetic resonance measurement of cobalt-substituted deuterolysin.
The substrate specificities of deuterolysin, a 19-kDa zinc-protease (EC 3.4.24.39) from Aspergillus oryzae, were investigated at pH 9.0 with various fluorogenic acyl-peptide-4-methylcoumaryl-7-amides (peptide-MCAs). N-Butoxycarbonyl-Arg-Val-Arg-Arg-MCA was the best substrate for deuterolysin. We therefore measured its kinetic parameters. Deuterolysin had high activity toward the peptide bonds next to pairs of basic residues in calf thymus histone H4. The specificity of cobalt-substituted deuterolysin (Co-deuterolysin) for peptide-MCAs was similar to that of native deuterolysin. The CD spectrum of Co-deuterolysin was similar to that of the native deuterolysin. The metal coordination sphere of Co-deuterolysin was analyzed by Q-band (33.9570 GHz) electron paramagnetic resonance (EPR) spectroscopy. Using computer simulation of EPR, we found the g principal values to be g(xx) = 5.20, g(yy) = 4.75, and g(zz) = 2.24; the metal center was a divalent cobalt ion in a high spin state.
Department of Bioengineering, Graduate School of Engineering, Soka University, Hachioji, Tokyo 192-8577, Japan.
Yuko
Y
Doi
Byung Rho
BR
Lee
Masamichi
M
Ikeguchi
Yasunori
Y
Ohoba
Tadaaki
T
Ikoma
Shozo
S
Tero-Kubota
Seigo
S
Yamauchi
Koji
K
Takahashi
Eiji
E
Ichishima
D000577:Amides / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D024361:Amino Acids, Basic / Q000378:metabolism; D000818:Animals; D001236:Aspergillus oryzae / Q000201:enzymology*; D001665:Binding Sites; D002417:Cattle; D002942:Circular Dichroism; D003035:Cobalt / Q000737:chemistry* / Q000378:metabolism; D003198:Computer Simulation; D004578:Electron Spin Resonance Spectroscopy; D004795:Enzyme Stability; D006657:Histones / Q000378:metabolism; D006358:Hot Temperature; D007700:Kinetics; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D013379:Substrate Specificity; D013950:Thymus Gland / Q000737:chemistry; D015032:Zinc / Q000378:metabolism*
D000577:Amides; D024361:Amino Acids, Basic; D006657:Histones; D003035:Cobalt; D008666:Metalloendopeptidases; D015032:Zinc