Kinetic and structural studies of the role of the active site residue Asp235 of human pyridoxal kinase

Biochem Biophys Res Commun. 2009 Mar 27;381(1):12-5. doi: 10.1016/j.bbrc.2009.01.170. Epub 2009 Feb 4.

Abstract

Pyridoxal kinase catalyzes the phosphorylation of pyridoxal (PL) to pyridoxal 5'-phosphate (PLP). A D235A variant shows 7-fold and 15-fold decreases in substrate affinity and activity, respectively. A D235N variant shows approximately 2-fold decrease in both PL affinity and activity. The crystal structure of D235A (2.5 A) shows bound ATP, PL and PLP, while D235N (2.3 A) shows bound ATP and sulfate. These results document the role of Asp235 in PL kinase activity. The observation that the active site of PL kinase can accommodate both ATP and PLP suggests that formation of a ternary Enz.PLP.ATP complex could occur in the wild-type enzyme, consistent with severe MgATP substrate inhibition of PL kinase in the presence of PLP.

Publication types

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

MeSH terms

  • Aspartic Acid / chemistry*
  • Aspartic Acid / genetics
  • Catalysis
  • Catalytic Domain
  • Crystallography, X-Ray
  • Humans
  • Kinetics
  • Mutagenesis
  • Phosphorylation
  • Protein Conformation
  • Protein Folding
  • Pyridoxal Kinase / chemistry*
  • Pyridoxal Kinase / genetics

Substances

  • Aspartic Acid
  • Pyridoxal Kinase