Purification and characterization of a cationic isoperoxidase from scented-geranium.
A strongly cationic isoperoxidase named PC3 was purified to homogeneity from scented-geranium (Pelargonium graveolens) callus by using DEAE-Sephacel chromatography, CM-cellulose chromatography and Sephacryl S-200 gel filtration, respectively. The enzyme was a glycoprotein with M(r) of ca. 58 kDa estimated by SDS-PAGE and Sephadex G-150 gel filtration. The pI value of the enzyme was 9.1. Kinetic studies revealed that PC3 had a very low K(m) value for scopoletin of 0.01 mM and could use ascorbate as a substrate. Interestingly, PC3 could not oxidize ferulic acid as a substrate. Chemical modification of the enzyme showed that PC3 was rapidly inactivated by His, Cys, Trp and Lys-specific reagents. The second order rate constants and reaction orders with respect to these inactivations were determined. Notably, ca. 4-7-fold activity boosting of PC3 occurred by adenine and imidazole when anilino substrates, such as o-dianisidine and o-phenylenediamine were oxidized, whereas this activity boosting did not occur when several phenolic substrates were used.
Lee
S H
SH
Department of Life Science, College of Science, Soonchunhyang University, Asan, 336-745, Choongnam, South Korea.
Kim
E S
ES
Lee
M Y
MY
D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D007700:Kinetics; D019684:Magnoliopsida / Q000201:enzymology*; D008970:Molecular Weight; D010544:Peroxidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity
D010544:Peroxidases; C445734:cationic isoperoxidase PC3