Allosteric interactions within subsites of a monomeric enzyme: kinetics of fluorogenic substrates of PI-specific phospholipase C.
Two novel water-soluble fluorescein myo-inositol phosphate (FLIP) substrates, butyl-FLIP and methyl-FLIP, were used to examine the kinetics and subsite interactions of Bacillus cereus phosphatidylinositol-specific phospholipase C. Butyl-FLIP exhibited sigmoidal kinetics when initial rates are plotted versus substrate concentration. The data fit a Hill coefficient of 1.2-1.5, suggesting an allosteric interaction between two sites. Two substrate molecules bind to this enzyme, one at the active site and one at a subsite, causing an increase in activity. The kinetic behavior is mathematically similar to that of well-known cooperative multimeric enzymes even though this phosphatidylinositol-specific phospholipase C is a small, monomeric enzyme. The less hydrophobic substrate, methyl-FLIP, binds only to the active site and not the activator site, and thus exhibits standard hyperbolic kinetics. An analytical expression is presented that accounts for the kinetics of both substrates in the absence and presence of a nonsubstrate short-chain phospholipid, dihexanoylphosphatidylcholine. The fluorogenic substrates detect activation at much lower concentrations of dihexanoylphosphatidylcholine than previously reported.
Birrell
G Bruce
GB
Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, OR 97403, USA.
Zaikova
Tatiana O
TO
Rukavishnikov
Aleksey V
AV
Keana
John F W
JF
Griffith
O Hayes
OH
D000495:Allosteric Site; D001409:Bacillus cereus / Q000737:chemistry / Q000201:enzymology; D001665:Binding Sites; D003198:Computer Simulation; D004789:Enzyme Activation; D019793:Fluorescein; D007295:Inositol Phosphates / Q000737:chemistry*; D007700:Kinetics; D008956:Models, Chemical; D010713:Phosphatidylcholines / Q000737:chemistry*; D043265:Phosphatidylinositol Diacylglycerol-Lyase / Q000737:chemistry*; D054801:Phosphoinositide Phospholipase C; D011485:Protein Binding; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013379:Substrate Specificity
C092445:1,2-dihexanoyl-sn-glycero-3-phosphorylcholine; D007295:Inositol Phosphates; D010713:Phosphatidylcholines; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D019793:Fluorescein