Whole-protein alanine-scanning mutagenesis of allostery: A large percentage of a protein can contribute to mechanism

Hum Mutat. 2017 Sep;38(9):1132-1143. doi: 10.1002/humu.23231. Epub 2017 Jun 16.


Many studies of allosteric mechanisms use limited numbers of mutations to test whether residues play "key" roles. However, if a large percentage of the protein contributes to allosteric function, mutating any residue would have a high probability of modifying allostery. Thus, a predicted mechanism that is dependent on only a few residues could erroneously appear to be supported. We used whole-protein alanine-scanning mutagenesis to determine which amino acid sidechains of human liver pyruvate kinase (hL-PYK; approved symbol PKLR) contribute to regulation by fructose-1,6-bisphosphate (Fru-1,6-BP; activator) and alanine (inhibitor). Each nonalanine/nonglycine residue of hL-PYK was mutated to alanine to generate 431 mutant proteins. Allosteric functions in active proteins were quantified by following substrate affinity over a concentration range of effectors. Results show that different residues contribute to the two allosteric functions. Only a small fraction of mutated residues perturbed inhibition by alanine. In contrast, a large percentage of mutated residues influenced activation by Fru-1,6-BP; inhibition by alanine is not simply the reverse of activation by Fru-1,6-BP. Moreover, the results show that Fru-1,6-BP activation would be extremely difficult to elucidate using a limited number of mutations. Additionally, this large mutational data set will be useful to train and test computational algorithms aiming to predict allosteric mechanisms.

Keywords: PKLR; alanine-scanning mutagenesis; allosteric mechanism; allosteric regulation; allostery; liver pyruvate kinase.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Alanine / pharmacology*
  • Algorithms
  • Allosteric Regulation
  • Computational Biology
  • Crystallography, X-Ray
  • Enzyme Activation
  • Fructosediphosphates / pharmacology*
  • Humans
  • Models, Molecular
  • Mutation*
  • Pyruvate Kinase / chemistry*
  • Pyruvate Kinase / genetics*
  • Pyruvate Kinase / metabolism


  • Fructosediphosphates
  • Pyruvate Kinase
  • fructose-1,6-diphosphate
  • Alanine