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

Hiromu Takematsu, Takehiro Kawano, Susumu Koyama, Yasunori Kozutsumi, Akemi Suzuki, Toshisuke Kawasaki

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Abstract

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.

Original languageEnglish
Pages (from-to)381-386
Number of pages6
JournalJournal of Biochemistry
Volume115
Issue number3
DOIs
Publication statusPublished - 03-1994
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

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