Studies of the Structure of Fructose-6-phosphate 2-Kinase:Fructose-2,6-bisphosphatase

Ryuzo Sakakibara, Tatsuya Tanaka, Kosaku Uyeda, E. Glen Richards, Helmut Thomas, Kenji Kangawa, Hisayuki Matsuo

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Abstract

Some physicochemical properties of a homogeneous preparation of a bifunctional enzyme, fructose-6-phosphate 2-kinase:fructose-2,6-bisphosphatase, were determined. The molecular weight of the enzyme is 101 000 as determined by high-speed sedimentation equilibrium. The molecular weight of dissociated enzyme is 55000 in 6 M guanidinium chloride by sedimentation equilibrium and in sodium dodecyl sulfate by polyacrylamide gel electrophoresis. A value of 4.7 was observed for the isoelectric point. Tryptic peptide maps and high-performance liquid chromatography of the trypsin-digested enzyme revealed approximately 60 peptides. Amino acid analysis of the enzyme shows that it contains 27 lysine and 36 arginine residues per 55 000 daltons. No free N-terminal amino acid residue was detectable, suggesting that it is blocked. Hydrolysis of the enzyme by carboxypeptidases A and B releases tyrosine followed by histidine and arginine, indicating that the amino acid sequence at the carboxyl terminus is probably -Arg-His-Tyr. Tryptic digestion of [32P]phosphofructose-6-phosphate 2-kinase:fructose-2,6-bisphosphatase yields a 32P-labeled peptide detected by tryptic peptide mapping and high-performance liquid chromatography. Thermolysin digestion of CNBr-cleaved 32P-enzyme also yields a single 32P-peptide. These results indicate thatf fructose-phosphate 2-kinase:fructose-2,6-bisphosphatase is a dimer of 55 000 daltons and the subunits are very similar, if not identical.

Original languageEnglish (US)
Pages (from-to)6818-6824
Number of pages7
JournalBiochemistry
Volume24
Issue number24
DOIs
StatePublished - Nov 1 1985

ASJC Scopus subject areas

  • Biochemistry

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    Sakakibara, R., Tanaka, T., Uyeda, K., Richards, E. G., Thomas, H., Kangawa, K., & Matsuo, H. (1985). Studies of the Structure of Fructose-6-phosphate 2-Kinase:Fructose-2,6-bisphosphatase. Biochemistry, 24(24), 6818-6824. https://doi.org/10.1021/bi00345a013