Effect of replacement of the amino and the carboxyl termini of rat testis fructose 6-phosphate, 2-kinase: Fructose 2,6-bisphosphatase with those of the liver and heart isozymes

Nobuaki Tominaga, Tomoyuki Tsujikawa, Yoshiko Minami, Ru Feng Wu, Fusao Watanabe, Ryuzo Sakakibara, Kosaku Uyeda

Research output: Contribution to journalArticle

8 Scopus citations

Abstract

Fru 6-P,2-kinase:Fru 2,6-Pase is a bifunctional enzyme, consisting of highly conserved catalytic domains and variable regulatory domains. The regulatory domains reside in either the N- or the C-terminus, depending upon the isozyme. The rat testis enzyme (RT2K) lacks the regulatory domain, but the rat liver and the bovine heart enzymes contain phosphorylation site(s) in the N- and the C-termini, respectively. In order to determine whether the regulatory domains can be swapped, we have constructed mutant enzymes in which the N- or the C-terminal tail of the testis enzyme was replaced with that of either the liver or the heart enzyme. The substitution with the N- terminus of the liver enzyme (RLN-RT2K) resulted in a small change in the kinetic properties of Fru 6-P,2-kinase, but that with the heart enzyme increased the K(Fru 6-P) 18-fold without affecting the V(max). The substitution with the C-terminus of the heart enzyme had little effect. The phosphorylation of RLN-RT2K increased K(Fru 6-P) fivefold as in the liver enzyme but did not affect the Fru 2,6-Pase, unlike the liver enzyme. All these mutant enzymes were more thermally labile than the wild type testis enzyme. RLN-RT2K was more sensitive to the denaturant. These results suggest that the N-terminus of the liver enzyme could interact with the kinase domain of the testis enzyme, regulating the kinase activity but was unable to affect the phosphatase domain. These differences could be explained by the large differences in net charges of the terminal tails.

Original languageEnglish (US)
Pages (from-to)275-281
Number of pages7
JournalArchives of Biochemistry and Biophysics
Volume347
Issue number2
DOIs
StatePublished - Nov 15 1997

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

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