Probing L-pyruvate kinase regulatory phosphorylation site by mutagenesis

Protein J. 2012 Oct;31(7):592-7. doi: 10.1007/s10930-012-9438-1.

Abstract

The activity of L-type pyruvate kinase (L-PK, ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) is regulated by phosphorylation of serine residue 12 of the N-terminal regulatory domain MEGPAGYLRR(10)AS ( 12 )VAQLTQEL(20)GTAFF of the protein. In this report we studied the effect of the point mutations around this phosphorylation site on the catalytic properties of this enzyme, by introducing amino acids A, L, K, Q and E into positions 9, 10 and 13 of this peptide sequence. It was found that some of these mutations in positions 9 and 10, although occurring at great distances from the enzyme's active site, affected the enzyme's activity by decreasing the effectiveness of phosphoenolpyruvate binding (PEP) with the enzyme, but had practically no influence on the binding effectiveness of the second substrate ADP. A similar asymmetric effect on the binding of these substrates was previously observed after phosphorylation of the enzyme regulatory N-domain peptide, and also after proteolytic truncation of the same N-terminal part of L-PK. All these results could be explained by the internal complex formation between the N-domain peptide and the enzyme's main body. The present study delineated the specificity of the internal binding site and revealed the possibility that the regulatory effect could be modulated by selecting mutation sites and amino acids introduced into the N-terminal domain structure.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Diphosphate / chemistry
  • Adenosine Diphosphate / metabolism
  • Amino Acid Sequence
  • Amino Acid Substitution
  • Animals
  • Catalytic Domain
  • Kinetics
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Phosphoenolpyruvate / chemistry
  • Phosphoenolpyruvate / metabolism
  • Phosphorylation
  • Pyruvate Kinase / chemistry
  • Pyruvate Kinase / genetics
  • Pyruvate Kinase / metabolism*
  • Rats

Substances

  • Adenosine Diphosphate
  • Phosphoenolpyruvate
  • Pyruvate Kinase