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-glycolylneuraminic 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 65. In order to determine the origin of b₃ detected in the cytosolic fraction of mouse liver, the molecular forms of b₅ mRNA expressed in mouse liver were analyzed. The polymerase chain reaction, with mouse liver cDNA and specific primers for the respective forms of b₅ mRNA, detected only the mRNA encoding the microsomal form in mouse liver. These results suggest the possibility that b₅ involved in the ternary complex formation in the cytosolic fraction of mouse liver originates from the microsomal form, which was solubilized after its synthesis. Possible participation of the microsomal b₅ on the endoplasmic reticulum in the hydroxylation reaction was also suggested by the results of an in vitro reconstitution assay involving isolated microsomes.