Preparation and kinetic studies of immobilised yeast cytochrome c peroxidase.
Yeast cytochrome c peroxidase (CcP) was purified from baker's yeast and immobilised onto a nylon membrane. The kinetics of the soluble and immobilised forms of the enzyme were investigated for the catalysed oxidation of potassium ferrocyanide in the presence of H2O2 and m-chloroperoxybenzoic acid. The pH dependence of the two forms of the enzyme differed. Although both the soluble and the immobilised enzymes showed optimal activity at pH 6.2, a different kinetic behaviour was demonstrated. Both forms of the enzyme showed similar activity toward H2O2, although when m-chloroperoxybenzoic acid was replaced as the electron acceptor, the immobilised form of the enzyme had a reduced turnover number and an increased Km. The activation energy of immobilised CcP was greater in the presence of both H2O2 [16.6 kJ mol-1] and m-chloroperoxybenzoic acid [37.9 kJ mol-1] than for soluble CcP [11.4 and 23.4 kJ mol-1, respectively]. The activities of both soluble and immobilised CcP were greatly reduced above 45 degrees C, although at higher temperatures the immobilised enzyme retained a relatively greater percentage of its maximum activity.
Cooper
J M
JM
Department of Clinical Biochemistry, Medical School, University of Newcastle upon Tyne, United Kingdom.
McNeil
C J
CJ
Bannister
J V
JV
D002384:Catalysis; D002723:Chlorobenzoates / Q000737:chemistry; D003578:Cytochrome-c Peroxidase / Q000737:chemistry / Q000302:isolation & purification*; D004789:Enzyme Activation; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000302:isolation & purification*; D005295:Ferrocyanides / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents / Q000737:chemistry; D007700:Kinetics; D010084:Oxidation-Reduction; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012995:Solubility; D013696:Temperature
D002723:Chlorobenzoates; D004800:Enzymes, Immobilized; D005295:Ferrocyanides; D007202:Indicators and Reagents; D006861:Hydrogen Peroxide; D003578:Cytochrome-c Peroxidase; C020354:hexacyanoferrate II; C000433:3-chloroperbenzoic acid