The stereoselective recognition of substrates by phosphoinositide kinases. Studies using synthetic stereoisomers of dipalmitoyl phosphatidylinositol.
Soluble phosphatidylinositol (PtdIns) 4- and 3-kinase activities were partially purified and characterized from human placental extracts. The placental PtdIns 4-kinase (type 3) has a Km for ATP of 460 microM and is kinetically different to a partially purified human erythrocyte, membrane-bound, PtdIns 4-kinase (type 2). These three inositol lipid kinases were then used to compare their substrate specificities against the four synthetic stereoisomers of dipalmitoyl PtdIns. Only the placental 4-kinase was influenced by the chirality of the glycerol moiety of PtdIns. However, neither of the 4-kinases was able to phosphorylate L-PtdIns and, therefore, these kinases have an absolute requirement for the inositol ring to be linked to the glyceryl backbone of the lipid through the D-1 position. Phosphoinositide 3-kinase, on the other hand, was found to phosphorylate both D- and L-PtdIns. While the 3-kinase phosphorylated exclusively the D-3 position of D-PtdIns, further analyses demonstrated that the same enzyme phosphorylated two sites on L-PtdIns, namely the D-6 and D-5 positions of the inositol ring. Some implications of these findings are discussed.
Macphee
C H
CH
SmithKline Beecham Pharmaceuticals, Welwyn, Hertfordshire, United Kingdom.
Carter
A N
AN
Ruiz-Larrea
F
F
Ward
J G
JG
Young
R C
RC
Downes
C P
CP
D019870:1-Phosphatidylinositol 4-Kinase; D000255:Adenosine Triphosphate / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D002855:Chromatography, Thin Layer; D005260:Female; D006801:Humans; D007700:Kinetics; D019869:Phosphatidylinositol 3-Kinases; D010716:Phosphatidylinositols / Q000378:metabolism; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism*; D010920:Placenta / Q000201:enzymology; D011247:Pregnancy; D013237:Stereoisomerism; D013379:Substrate Specificity
D010716:Phosphatidylinositols; C074858:dipalmitoyl phosphatidylinositol; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D019869:Phosphatidylinositol 3-Kinases; D019870:1-Phosphatidylinositol 4-Kinase