Reactions of soybean peroxidase and hydrogen peroxide pH 2.4-12.0, and veratryl alcohol at pH 2.4.
Peroxidase from soybean seed coat (SBP) has properties that makes it particularly suited for practical applications. Therefore, it is essential to know its fundamental enzymatic properties. Stopped-flow techniques were used to investigate the pH dependence of the reaction of SBP and hydrogen peroxide. The reaction is linearly dependent on hydrogen peroxide concentration at acidic and neutral pH with the second order rate constant k(1)=2.0x10(7) M(-1) s(-1), pH 4-8. From pH 9.3 to 10.2 the reaction is biphasic, a novel observation for a peroxidase at alkaline pH. A fast reaction has the characteristics of the reaction at neutral pH, and a slow reaction shows hyperbolic dependence on hydrogen peroxide concentration. At pH >10.5 only the slow reaction is seen. The shift in mechanism is coincident with the change in haem iron co-ordination to a six-coordinate low spin hydroxy ligated alkaline form. The pK(a) value for the alkaline transition was observed at 9.7+/-0.1, 9.6+/-0.1 and 9.9+/-0.2 by spectrophotometric titration, the fast phase amplitude, and decrease in the apparent second order rate constant, respectively. An acidic pK(a) at 3.2+/-0.3 was also determined from the apparent second order rate constant. The reactions of soybean peroxidase compounds I and II with veratryl alcohol at pH 2.44 give very similar second order rate constants, k(2)=(2.5+/-0.1)x10(4) M(-1) s(-1) and k(3)=(2.2+/-0.1)x10(4) M(-1) s(-1), respectively, which is unusual. The electronic absorption spectra of compounds I, II and III at pH 7.07 show characteristic bands at 400 and 651 nm (compound I), 416, 527 and 555 nm (compound II), and 414, 541 and 576 nm (compound III). No additional intermediates were observed.
Nissum
M
M
Department of Protein Chemistry, Institute of Molecular Biology, University of Copenhagen, Denmark.
Schiødt
C B
CB
Welinder
K G
KG
D001592:Benzyl Alcohols / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D010544:Peroxidases / Q000378:metabolism*; D012639:Seeds / Q000201:enzymology; D030262:Soybean Proteins / Q000378:metabolism; D013025:Glycine max / Q000201:enzymology*; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman
D001592:Benzyl Alcohols; D030262:Soybean Proteins; D006861:Hydrogen Peroxide; D010544:Peroxidases; C120494:anionic peroxidase; C042197:veratryl alcohol