Reaction mechanism underlying CMP-N-acetylneuraminic acid hydroxylation in mouse liver: formation of a ternary complex of cytochrome b5, CMP-N-acetylneuraminic acid, and a hydroxylation enzyme.
We have proposed that CMP-N-acetylneuraminic acid (CMP-NeuAc) hydroxylation is mediated by an electron transport system consisting of cytochrome b5 (b5), b5 reducing factor(s), and CMP-NeuAc hydroxylase, all of which have been detected in the cytosolic fraction of mouse liver [Kozutsumi, Y., Kawano, T., Yamakawa, T., & Suzuki, A. (1990) J. Biochem. 108, 704-706]. In order to elucidate the reaction mechanism underlying CMP-NeuAc hydroxylation, the interaction between b5 and the hydroxylase was studied using a b5-immobilized affinity column. The enzyme activity was retarded on the b5 column in the presence of the substrate, CMP-NeuAc, but not in the presence of the reaction product, CMP-N-glycolyneuraminic acid (CMP-NeuGc). These findings suggest that the binding of CMP-NeuAc to CMP-NeuAc hydroxylase changes the conformation of the enzyme so as to construct a recognition site for b5, followed by the formation of a ternary complex through this domain. Then the transport of electrons from NAD(P)H to the enzyme through b5 takes place, CMP-NeuAc is converted to CMP-NeuGc, and finally the ternary complex dissociates into its components to release CMP-NeuGc. It is known that a soluble form of b5 is abundant in erythrocytes and is synthesized from a mRNA different from that for the microsomal form of b5. In order to determine the origin of b5 detected in the cytosolic fraction of mouse liver, the molecular forms of b5 mRNA expressed in mouse liver were analyzed.(ABSTRACT TRUNCATED AT 250 WORDS)
Takematsu
H
H
Department of Biological Chemistry, Faculty of Pharmaceutical Sciences, Kyoto University.
Kawano
T
T
Koyama
S
S
Kozutsumi
Y
Y
Suzuki
A
A
Kawasaki
T
T
D000818:Animals; D001483:Base Sequence; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid / Q000378:metabolism*; D015786:Cytochromes b5 / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D006900:Hydroxylation; D051379:Mice; D008807:Mice, Inbred BALB C; D008862:Microsomes, Liver / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism
D018076:DNA, Complementary; D012333:RNA, Messenger; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid; D015786:Cytochromes b5; D006899:Mixed Function Oxygenases; C074383:CMPacetylneuraminate monooxygenase