Crystal structure of the H256A mutant of rat testis fructose-6- phosphate,2-kinase/fructose-2,6-bisphosphatase: Fructose 6-phosphate in the active site leads to mechanisms for both mutant and wild type bisphosphatase activities

Mi H. Yuen, Hiroyuki Mizuguchi, Yong Hwan Lee, Paul F. Cook, Kosaku Uyeda, Charles A. Hasemann

Research output: Contribution to journalArticle

32 Citations (Scopus)

Abstract

Fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase (Fru-6-P,2- kinase/Fru-2,6-Pase) is a bifunctional enzyme, catalyzing the interconversion of β-D-fructose-6-phosphate (Fru-6-P) and fructose-2,6-bisphosphate (Fru- 2,6-P2) at distinct active sites. A mutant rat testis isozyme with an alanine replacement for the catalytic histidine (H256A) in the Fru-2,6-Pase domain retains 17% of the wild type activity (Mizuguchi, H., Cook, P. F., Tai, C-H., Hasemann, C. A., and Uyeda, K. (1998) J. Biol. Chem. 274, 2166- 2175). We have solved the crystal structure of H256A to a resolution of 2.4 Å by molecular replacement. Clear electron density for Fru-6-P is found at the Fru-2,6-Pase active site, revealing the important interactions in substrate/product binding. A superposition of the H256A structure with the RT2K-Wo structure reveals no significant reorganization of the active site resulting from the binding of Fru-6-P or the H256A mutation. Using this superposition, we have built a view of the Fru-2,6-P2-bound enzyme and identify the residues responsible for catalysis. This analysis yields distinct catalytic mechanisms for the wild type and mutant proteins. The wild type mechanism would lead to an inefficient transfer of a proton to the leaving group Fru-6-P, which is consistent with a view of this event being rate-limiting, explaining the extremely slow turnover (0.032 s-1) of the Fru-2,6-Pase in all Fru-6-P,2-kinase/Fru-2,6-Pase isozymes.

Original languageEnglish (US)
Pages (from-to)2176-2184
Number of pages9
JournalJournal of Biological Chemistry
Volume274
Issue number4
DOIs
StatePublished - Jan 22 1999

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Phosphofructokinase-2
Testis
Rats
Catalytic Domain
Phosphotransferases
Crystal structure
Isoenzymes
Mutant Proteins
Enzymes
Catalysis
Histidine
Alanine
Carrier concentration
fructose-6-phosphate
Protons
Binding Sites
Electrons
Mutation
Substrates

ASJC Scopus subject areas

  • Biochemistry

Cite this

Crystal structure of the H256A mutant of rat testis fructose-6- phosphate,2-kinase/fructose-2,6-bisphosphatase : Fructose 6-phosphate in the active site leads to mechanisms for both mutant and wild type bisphosphatase activities. / Yuen, Mi H.; Mizuguchi, Hiroyuki; Lee, Yong Hwan; Cook, Paul F.; Uyeda, Kosaku; Hasemann, Charles A.

In: Journal of Biological Chemistry, Vol. 274, No. 4, 22.01.1999, p. 2176-2184.

Research output: Contribution to journalArticle

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title = "Crystal structure of the H256A mutant of rat testis fructose-6- phosphate,2-kinase/fructose-2,6-bisphosphatase: Fructose 6-phosphate in the active site leads to mechanisms for both mutant and wild type bisphosphatase activities",
abstract = "Fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase (Fru-6-P,2- kinase/Fru-2,6-Pase) is a bifunctional enzyme, catalyzing the interconversion of β-D-fructose-6-phosphate (Fru-6-P) and fructose-2,6-bisphosphate (Fru- 2,6-P2) at distinct active sites. A mutant rat testis isozyme with an alanine replacement for the catalytic histidine (H256A) in the Fru-2,6-Pase domain retains 17{\%} of the wild type activity (Mizuguchi, H., Cook, P. F., Tai, C-H., Hasemann, C. A., and Uyeda, K. (1998) J. Biol. Chem. 274, 2166- 2175). We have solved the crystal structure of H256A to a resolution of 2.4 {\AA} by molecular replacement. Clear electron density for Fru-6-P is found at the Fru-2,6-Pase active site, revealing the important interactions in substrate/product binding. A superposition of the H256A structure with the RT2K-Wo structure reveals no significant reorganization of the active site resulting from the binding of Fru-6-P or the H256A mutation. Using this superposition, we have built a view of the Fru-2,6-P2-bound enzyme and identify the residues responsible for catalysis. This analysis yields distinct catalytic mechanisms for the wild type and mutant proteins. The wild type mechanism would lead to an inefficient transfer of a proton to the leaving group Fru-6-P, which is consistent with a view of this event being rate-limiting, explaining the extremely slow turnover (0.032 s-1) of the Fru-2,6-Pase in all Fru-6-P,2-kinase/Fru-2,6-Pase isozymes.",
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T1 - Crystal structure of the H256A mutant of rat testis fructose-6- phosphate,2-kinase/fructose-2,6-bisphosphatase

T2 - Fructose 6-phosphate in the active site leads to mechanisms for both mutant and wild type bisphosphatase activities

AU - Yuen, Mi H.

AU - Mizuguchi, Hiroyuki

AU - Lee, Yong Hwan

AU - Cook, Paul F.

AU - Uyeda, Kosaku

AU - Hasemann, Charles A.

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AB - Fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase (Fru-6-P,2- kinase/Fru-2,6-Pase) is a bifunctional enzyme, catalyzing the interconversion of β-D-fructose-6-phosphate (Fru-6-P) and fructose-2,6-bisphosphate (Fru- 2,6-P2) at distinct active sites. A mutant rat testis isozyme with an alanine replacement for the catalytic histidine (H256A) in the Fru-2,6-Pase domain retains 17% of the wild type activity (Mizuguchi, H., Cook, P. F., Tai, C-H., Hasemann, C. A., and Uyeda, K. (1998) J. Biol. Chem. 274, 2166- 2175). We have solved the crystal structure of H256A to a resolution of 2.4 Å by molecular replacement. Clear electron density for Fru-6-P is found at the Fru-2,6-Pase active site, revealing the important interactions in substrate/product binding. A superposition of the H256A structure with the RT2K-Wo structure reveals no significant reorganization of the active site resulting from the binding of Fru-6-P or the H256A mutation. Using this superposition, we have built a view of the Fru-2,6-P2-bound enzyme and identify the residues responsible for catalysis. This analysis yields distinct catalytic mechanisms for the wild type and mutant proteins. The wild type mechanism would lead to an inefficient transfer of a proton to the leaving group Fru-6-P, which is consistent with a view of this event being rate-limiting, explaining the extremely slow turnover (0.032 s-1) of the Fru-2,6-Pase in all Fru-6-P,2-kinase/Fru-2,6-Pase isozymes.

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