Pyruvate:NADP+ oxidoreductase from Euglena gracilis: mechanism of O2-inactivation of the enzyme and its stability in the aerobe.
O2-inactivation of pyruvate:NADP+ oxidoreductase from mitochondria of Euglena gracilis was studied in vitro, and a mechanism which consists of two sequential stages was proposed. Initially, the enzyme is inactivated by the direct action of O2 in a process obeying second-order kinetics. Although the catalytic activity for pyruvate oxidation is lost by this initial inactivation, NADPH oxidation with artificial electron acceptors still occurs. Subsequently, a secondary, O2-independent inactivation occurs, rendering the enzyme completely inactive. Pyruvate stimulates the O2-inactivation while CoA and NADP+ protect the enzyme from O2. The O2-inactivation is accelerated by reduction of the enzyme with pyruvate and CoA. Reactivation of the O2-inactivated enzyme was studied in Ar by incubation with Fe2+ in the presence of some other reducing reagent such as dithiothreitol. The evidence obtained indicates that the partially inactivated enzyme, which retains catalytic activity for NADPH oxidation, can be reactivated, but the completely inactivated enzyme is not. When Euglena cells were exposed to 100% O2 the enzyme in the cells was inactivated by O2, but the rate was quite slow compared with that observed in vitro. The enzyme inactivated by O2 in the cells was almost completely reactivated in vitro by incubation with Fe2+ and other reducing reagents in Ar, suggesting that the secondary, O2-independent inactivation does not occur in situ. When the cells were returned to air, reactivation of the O2-inactivated enzyme in the cells began immediately. The enzyme, kept in isolated, intact mitochondria, was stable in air; however, the enzyme was inactivated by O2 when the mitochondria were incubated with a high concentration of pyruvate.
Inui
H
H
Department of Food and Nutrition, Narabunka Women's Junior College, Nara, Japan.
Miyatake
K
K
Nakano
Y
Y
Kitaoka
S
S
D000818:Animals; D004789:Enzyme Activation; D004795:Enzyme Stability; D005056:Euglena gracilis / Q000201:enzymology*; D007658:Ketone Oxidoreductases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology; D010100:Oxygen / Q000494:pharmacology*
D007658:Ketone Oxidoreductases; C053164:pyruvate dehydrogenase (NADP+); D010100:Oxygen