Mg2+ induces conformational changes in the catalytic subunit of phosphorylase kinase, whether by itself or as part of the holoenzyme complex

Deborah A. Wilkinson, Thomas J. Fitzgerald, Tony Marion, Gerald M. Carlson

Research output: Contribution to journalArticle

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Abstract

Phosphorylase kinase (PhK) from skeletal muscle is a structurally complex, highly regulated, hexadecameric enzyme of subunit composition (αβγδ)4. Previous studies have revealed that the activity of its catalytic γ subunit is controlled by alterations in quaternary structure initiated at allosteric and covalent modification sites on PhK's three regulatory subunits; however, changes in the conformation of the holoenzyme initiated by the catalytic subunit have been more difficult to document. In this study a monoclonal antibody (mAb γ79) has been generated against isolated γ subunit and used as a conformational probe of that subunit. The epitope recognized by this antibody is within the catalytic core of the γ subunit, between residues 100 and 240, and monovalent fragments of the antibody inhibit the catalytic activity of the holoenzyme, the γ-calmodulin binary complex, and the free γ subunit. Activation of PhK by a variety of mechanisms known or thought to act through its regulatory subunits (phosphorylation, ADP binding, or alkaline pH) increased the binding of the holoenzyme to immobilized mAb γ79, indicating that activation by any of these distinct mechanisms involves repositioning of the portion of the catalytic domain of the γ subunit containing the epitope for mAb γ79. The activating ligand Mg2+ also stimulated the binding of the PhK holoenzyme to immobilized mAb γ79, as well as the binding of mAb γ79 to immobilized γ subunit. Thus, Mg2+ increases the accessibility of the mAb γ79 epitope in both the isolated γ subunit and in the holoenzyme. Our results suggest that previously reported influences of Mg2+ on the quaternary structure of the PhK holoenzyme are directly mediated by the γ subunit.

Original languageEnglish (US)
Pages (from-to)157-164
Number of pages8
JournalJournal of Protein Chemistry
Volume18
Issue number2
DOIs
StatePublished - May 19 1999

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Phosphorylase Kinase
Epitopes
Holoenzymes
Catalytic Domain
Antibodies
Chemical activation
Calmodulin
Administrative data processing
Phosphorylation
Monoclonal antibodies
Muscle
Conformations
Catalyst activity
Enzymes
Catalytic Antibodies
Ligands
Immunoglobulin Fragments
Chemical analysis
Adenosine Diphosphate
Skeletal Muscle

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Mg2+ induces conformational changes in the catalytic subunit of phosphorylase kinase, whether by itself or as part of the holoenzyme complex. / Wilkinson, Deborah A.; Fitzgerald, Thomas J.; Marion, Tony; Carlson, Gerald M.

In: Journal of Protein Chemistry, Vol. 18, No. 2, 19.05.1999, p. 157-164.

Research output: Contribution to journalArticle

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