Human platelet 12-lipoxygenase: naturally occurring Q261/R261 variants and N544L mutant show altered activity but unaffected substrate binding and membrane association behavior.
The single nucleotide polymorphism (SNP) R261Q in the human platelet 12-lipoxygenase has been correlated with several human diseases. To understand better the biological performance we have compared enzymatic properties of the recombinant enzymes: 'wild-type' as Q261 and R261 variants with a single Q261R mutation at the enzyme periphery and N544L mutant with an altered active site. The R261 variant does not follow the same kinetics such as WT-Q261 showing a lag phase, a slower accumulation of product, following a different time-course without reaching plateau characteristic for the Q261 variant. The N544L substitution in the active site almost eradicates enzymatic activity proving that asparagine is as important for catalysis as the conserved histidines and C-terminal isoleucine. All three enzymes have comparable substrate binding and membrane association behavior. We conclude that the naturally occurring SNP, causing single mutation at a location distant to the active site, can alter the protein-protein association of this oligomeric enzyme making impact on kinetic properties of an allosteric mechanism and molecular recognition/signaling at a submembrane frontier.
Aleem
Ansari M
AM
Urology Research Center, College of Medicine, University of Toledo, Toledo, OH 43614, USA.
Wells
Leigh
L
Jankun
Jerzy
J
Walther
Matthias
M
Kühn
Hartmut
H
Reinartz
Jeannette
J
Skrzypczak-Jankun
Ewa
E
D000495:Allosteric Site; D001092:Arachidonate 12-Lipoxygenase / Q000097:blood / Q000737:chemistry / Q000235:genetics*; D001792:Blood Platelets / Q000201:enzymology*; D002462:Cell Membrane / Q000378:metabolism; D004795:Enzyme Stability; D006801:Humans; D008958:Models, Molecular; D020641:Polymorphism, Single Nucleotide; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D011994:Recombinant Proteins; D001092:Arachidonate 12-Lipoxygenase