Structural and functional linkages between subunit interfaces in mammalian pyruvate kinase

John O. Wooll, Robert H E Friesen, Mark White, Stanley Watowich, Robert O. Fox, James Lee, Edmund W. Czerwinski

Research output: Contribution to journalArticle

37 Citations (Scopus)

Abstract

Mammalian pyruvate kinase (PK) is a four-domain enzyme that is active as a homo-tetramer. Tissue-specific isozymes of PK exhibit distinct levels of allosteric regulation. PK expressed in muscle tissue (M1-PK)shows hyperbolic steady-state kinetics, whereas PK expressed in kidney tissue (M2-PK)displays sigmoidal kinetics. Rabbit M1 and M2-PK are isozymes whose sequences differ in only 22 out of 530 residues per subunit, and these changes are localized in an inter-subunit interface. Previous studies have shown that a single amino acid mutation to M1-PK at either the Y (S402P) or Z (T340 M) subunit interface can confer a level of allosteric regulation that is intermediate to M1-PK and M2-PK. In an effort to elucidate the roles of the inter-subunit interaction in signal transmission and the functional/structural connectivity between these interfaces, the S402P mutant of M1-PK was crystallized and its structure resolved to 2.8 Å. Although the overall S402P M1-PK structure is nearly identical with the wild-type structure within experimental error, significant differences in the conformation of the backbone are found at the site of mutation along the Y interface. In addition, there is a significant change along the Z interface, namely, a loss of an inter-subunit salt-bridge between Asp177 of domain B and Arg341 of domain A of the opposing subunit. Concurrent with the loss of the salt-bridge is an increase in the degree of rotational flexibility of domain B that constitutes the active site. Comparison of previous PK structures shows a correlation between an increase in this domain movement with the loss of the Asp177: Arg341 salt-bridge. These results identify the structural linkages between the Y and Z interfaces in regulating the interconversion of conformational states of rabbit M1-PK.

Original languageEnglish (US)
Pages (from-to)525-540
Number of pages16
JournalJournal of Molecular Biology
Volume312
Issue number3
DOIs
StatePublished - Sep 21 2001

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Pyruvate Kinase
Allosteric Regulation
Salts
Rabbits
Mutation
Isoenzymes
Catalytic Domain

Keywords

  • Allosterism
  • Pyruvate kinase
  • Structure
  • Subunit communication
  • X-ray crystallography

ASJC Scopus subject areas

  • Virology

Cite this

Structural and functional linkages between subunit interfaces in mammalian pyruvate kinase. / Wooll, John O.; Friesen, Robert H E; White, Mark; Watowich, Stanley; Fox, Robert O.; Lee, James; Czerwinski, Edmund W.

In: Journal of Molecular Biology, Vol. 312, No. 3, 21.09.2001, p. 525-540.

Research output: Contribution to journalArticle

Wooll, John O. ; Friesen, Robert H E ; White, Mark ; Watowich, Stanley ; Fox, Robert O. ; Lee, James ; Czerwinski, Edmund W. / Structural and functional linkages between subunit interfaces in mammalian pyruvate kinase. In: Journal of Molecular Biology. 2001 ; Vol. 312, No. 3. pp. 525-540.
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