Structure
Volume 6, Issue 2, 15 February 1998, Pages 195-210
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Research Article
The allosteric regulation of pyruvate kinase by fructose-1,6-bisphosphate

https://doi.org/10.1016/S0969-2126(98)00021-5Get rights and content
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Abstract

Background: Yeast pyruvate kinase (PK) catalyzes the final step in glycolysis. The enzyme therefore represents an important control point and is allosterically activated by fructose-1,6-bisphosphate (FBP). In mammals the enzyme is found as four different isozymes with different regulatory properties: two of these isozymes are produced by alternate splicing. The allosteric regulation of PK is directly related to proliferation of certain cell types, as demonstrated by the expression of an allosterically regulated isozyme in tumor cells. A model for the allosteric transition from the inactive (T) state to the active (R) state has been proposed previously, but until now the FBP-binding site had not been identified.

Results: We report here the structures of PK from yeast complexed with a substrate analog and catalytic metal ions in the presence and absence of bound FBP. The allosteric site is located 40 å from the active site and is entirely located in the enzyme regulatory (C) domain. A phosphate-binding site for the allosteric activator is created by residues encoded by a region of the gene corresponding to the alternately spliced exon of mammalian isozymes. FBP activation appears to induce several conformational changes among active-site sidechains through a mechanism that is most likely to involve significant domain motions, as previously hypothesized.

Conclusions: The structure and location of the allosteric activator site agrees with the pattern of alternate genetic splicing of the PK gene in multicellular eukaryotes that distinguishes between a non-regulated isozyme and the regulated fetal isozymes. The conformational differences observed between the active sites of inactive and fully active PK enzymes is in agreement with the recently determined thermodynamic mechanism of allosteric activation through a ‘metal relay’ that increases the affinity of the enzyme for its natural phosphoenolpyruvate substrate.

Keywords

allostery
alternative splicing
isczyme shift
pyruvate kinase

Cited by (0)

MS Jurica, Molecular and Cellular Biology Program of the University of Washington and Fred Hutchinson Cancer Research Center A3-023, 1124 Columbia Street, Seattle, WA 98104, USA.

A Mesecar, Department of Molecular and Cell Biology, Stanley Hall, University of California, Berkeley CA 94720, USA.

PJ Heath and BL Stoddard, Program in Structural Biology, Division of Basic Sciences, Fred Hutchinson Cancer Research Center A3-023, 1124 Columbia Street, Seattle, WA 98104, USA.

W Shi, Department of Biochemistry and Molecular Biology, Pennsylvania State University, 152 Davey Lab, University Park, PA 16802, USA.

T Nowak, Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA.

E-mail adddress for BL Stoddard (corresponding author): [email protected].