Biochem.J.; (1986) 233, 3, 493-97

Inactivation of 3-alpha-hydroxysteroid-dehydrogenase by superoxide radicals: modification of histidine and cysteine residues causes the conformational change

Kim-H-S; Minard-P; Legoy-M-D; Thomas-D

3-Alpha-hydroxysteroid-dehydrogenase (EC-1.1.1.50) from Pseudomonas testosteroni was inactivated by superoxide radicals generated by the aerobic xanthine-oxidase reaction. When superoxide-dismutase (EC-1.15.1.1) (SOD) was added at the time of initiation of the xanthine-oxidase reaction, inactivation was almost totally prevented by enzyme activity could not be completely recovered when SOD was added 2 min after the initiation. SOD prevented the inactivation of 3-alpha-HSDH completely at low concentrations of xanthine-oxidase (0.02 uM) but at high concentrations of xanthine-oxidase (0.2 uM), the inactivation was not completely prevented. NAD+, cattle serum albumin, and histidine and cysteine as free amino acids also partially protected the enzyme from inactivation. The NADH-binding properties were determined by fluorescence spectroscopy and no variation was found between the native enzyme and the unmodified fraction of the partly inactivated one. It is suggested that the modification of histidine and cysteine residues by superoxide radicals may cause the conformational change of the enzyme and the consequent loss of catalytic activity.