Biochem. J. (1977) 165, 81-87 Printed in Great Britain
81
A Pulse-Radiolysis Study of the Manganese-Containing Superoxide Dismutase from Bacillus stearothermophilus FURTHER STUDIES ON THE PROPERTIES OF THE ENZYME By MICHAEL E. McADAM, FRANCOIS LAVELLE,* RICHARD A. FOX and E. MARTIN FIELDEN Division ofPhysics, Institute of Cancer Research, Clifton Avenue, Sutton, Surrey SM2 5PX, U.K.
(Received 2 November 1976) In the preceding paper the mechanism of catalysis of the manganese-containing superoxide dismutase from Bacillus stearothermophilus was shown to involve a 'fast cycle' and a 'slow cycle' [McAdam, Fox, Lavelle & Fielden, 1977 (Biochem. J. 165, 71-79)]. Further properties of the enzyme are considered in the present paper. Pulse-radiolysis studies, under conditions of low substrate concentration to enzyme concentration (i.e. when the fast cycle predominates), showed that enzyme activity decreases as pH increases (6.5-10.2). Activity was unaffected by the addition of H202 or NaN3 but slightly decreased by KCN. Both H202 and the reducing radical anion CO2- caused a decrease in A480 of the native enzyme. The rate of the fast catalytic cycle was independent of temperature (5-55°C), and as temperature increases the slow catalytic cycle becomes relatively more important. Arrhenius parameters of the rate constants were estimated. The possible identity of the various forms of the enzyme is considered. In the preceding paper (McAdam et al., 1977), the essential kinetic features of the mechanism of action of Mn- (manganese-containing) superoxide dismutase from Bacillus stearothermophilus were described. In the present paper further observations on the properties of the enzyme are reported and, where useful, compared with the kinetic observations. Whenever such comparisons are made, the notation used will be that of Model I (McAdam et al., 1977):
EA + 02EB
+
02
-
1 > EB + 02 -EA+
H202
(1) (2)
EB+ 02-.
S3 > EC
(3)
Ec
> EA
(4)
Materials and Methods Experimental procedures and enzyme samples were as described previously (McAdam et al., 1977), and unless stated otherwise solutions studied by pulse radiolysis contained ethanol (0.1 M), sodium pyrophosphate (2mM) and EDTA (100puM). Results and Discussion Effect of composition of assay medium on measured activity For activity determination by the pulse-radiolysis method it is necessary to maintain a relatively high * Permanent address: Service de Biochimie-Physique, Institut de Biologie Physico-Chimique, 13 Rue Pierre et Marie Curie, 75005 Paris, France. Vol. 165
pH to decrease the spontaneous dismutation of 02- . It is also desirable to convert hydroxyl radicals, primary products of the radiolysis of water, into O2-.
These requirements demand a buffered medium containing ethanol or sodium formate (Fielden et al., 1974). When the ratio of substrate concentration to enzyme concentration ([O2-J/[Eo]) is low (
81
A Pulse-Radiolysis Study of the Manganese-Containing Superoxide Dismutase from Bacillus stearothermophilus FURTHER STUDIES ON THE PROPERTIES OF THE ENZYME By MICHAEL E. McADAM, FRANCOIS LAVELLE,* RICHARD A. FOX and E. MARTIN FIELDEN Division ofPhysics, Institute of Cancer Research, Clifton Avenue, Sutton, Surrey SM2 5PX, U.K.
(Received 2 November 1976) In the preceding paper the mechanism of catalysis of the manganese-containing superoxide dismutase from Bacillus stearothermophilus was shown to involve a 'fast cycle' and a 'slow cycle' [McAdam, Fox, Lavelle & Fielden, 1977 (Biochem. J. 165, 71-79)]. Further properties of the enzyme are considered in the present paper. Pulse-radiolysis studies, under conditions of low substrate concentration to enzyme concentration (i.e. when the fast cycle predominates), showed that enzyme activity decreases as pH increases (6.5-10.2). Activity was unaffected by the addition of H202 or NaN3 but slightly decreased by KCN. Both H202 and the reducing radical anion CO2- caused a decrease in A480 of the native enzyme. The rate of the fast catalytic cycle was independent of temperature (5-55°C), and as temperature increases the slow catalytic cycle becomes relatively more important. Arrhenius parameters of the rate constants were estimated. The possible identity of the various forms of the enzyme is considered. In the preceding paper (McAdam et al., 1977), the essential kinetic features of the mechanism of action of Mn- (manganese-containing) superoxide dismutase from Bacillus stearothermophilus were described. In the present paper further observations on the properties of the enzyme are reported and, where useful, compared with the kinetic observations. Whenever such comparisons are made, the notation used will be that of Model I (McAdam et al., 1977):
EA + 02EB
+
02
-
1 > EB + 02 -EA+
H202
(1) (2)
EB+ 02-.
S3 > EC
(3)
Ec
> EA
(4)
Materials and Methods Experimental procedures and enzyme samples were as described previously (McAdam et al., 1977), and unless stated otherwise solutions studied by pulse radiolysis contained ethanol (0.1 M), sodium pyrophosphate (2mM) and EDTA (100puM). Results and Discussion Effect of composition of assay medium on measured activity For activity determination by the pulse-radiolysis method it is necessary to maintain a relatively high * Permanent address: Service de Biochimie-Physique, Institut de Biologie Physico-Chimique, 13 Rue Pierre et Marie Curie, 75005 Paris, France. Vol. 165
pH to decrease the spontaneous dismutation of 02- . It is also desirable to convert hydroxyl radicals, primary products of the radiolysis of water, into O2-.
These requirements demand a buffered medium containing ethanol or sodium formate (Fielden et al., 1974). When the ratio of substrate concentration to enzyme concentration ([O2-J/[Eo]) is low (
