Enzymatic activation of autotaxin by divalent cations without EF-hand loop region involvement.
Autotaxin (ATX) is a recently described member of the nucleotide pyrophosphatase/phosphodiesterase (NPP) family of proteins with potent tumor cell motility-stimulating activity. Like other NPPs, ATX is a glycoprotein with peptide sequences homologous to the catalytic site of bovine intestinal alkaline phosphodiesterase (PDE) and the loop region of an EF-hand motif. The PDE active site of ATX has been associated with the motility-stimulating activity of ATX. In this study, we examined the roles of the EF-hand loop region and of divalent cations on the enzymatic activities of ATX. Ca(2+) or Mg(2+) was each demonstrated to increase the PDE activity of ATX in a concentration-dependent manner, whereas incubation of ATX with chelating agents abolished this activity, indicating a requirement for divalent cations. Non-linear regression analysis of enzyme kinetic data indicated that addition of these divalent cations increases reaction velocity predominantly through an effect on V(max.) Three mutant proteins, Ala(740)-, Ala(742)-, and Ala(751)-ATX, in the EF-hand loop region of ATX had enzymatic activity comparable to that of the wild-type protein. A deletion mutation of the entire loop region resulted in slightly reduced PDE activity but normal motility-stimulating activity. However, the PDE activity of this same deletion mutant remained sensitive to augmentation by cations, strongly implying that cations exert their effect by interactions outside of the EF-hand loop region.
Lee
J
J
College of Medicine, Konyang University, Nonsan 320-711, South Korea.
Jung
I D
ID
Nam
S W
SW
Clair
T
T
Jeong
E M
EM
Hong
S Y
SY
Han
J W
JW
Lee
H W
HW
Stracke
M L
ML
Lee
H Y
HY
D000595:Amino Acid Sequence; D000818:Animals; D001711:Biotransformation / Q000187:drug effects; D019556:COS Cells; D002413:Cations, Divalent / Q000494:pharmacology; D002614:Chelating Agents / Q000494:pharmacology; D005956:Glucose-6-Phosphate Isomerase / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D043264:Phosphodiesterase I; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011755:Pyrophosphatases / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D000099189:Lysophospholipase D
D002413:Cations, Divalent; D002614:Chelating Agents; D005956:Glucose-6-Phosphate Isomerase; D006023:Glycoproteins; D009097:Multienzyme Complexes; D043264:Phosphodiesterase I; D010727:Phosphoric Diester Hydrolases; D011755:Pyrophosphatases; D000099189:Lysophospholipase D; C405782:nucleotide pyrophosphatase - phosphodiesterase I