Phosphatidylinositol phospholipase C is activated allosterically by the aminoglycoside G418. 2-deoxy-2-fluoro-scyllo-inositol-1-O-dodecylphosphonate and its analogs inhibit glycosylphosphatidylinositol phospholipase C.
Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus is inhibited by myo-inositol-1-O-dodecylphosphonate (Ins-1-O-dodecylphosphonate) (Morris, J. C., Ping-Sheng, L., Shen, T. Y., and Mensa-Wilmot, K.(1995) J. Biol. Chem. 270, 2517-2524). A set of novel fluorinated 2-deoxy-Ins-1-O-dodecylphosphonates were tested against PI-PLC, with potent competitive inhibition by 2-deoxy-2-fluoro-scyllo-Ins-1-O-dodecylphosphonate (VP-616L) (Xi(50) = 0.09). 2-Deoxy-2-fluoro-myo-Ins-1-O-dodecylphosphonate and 2-deoxy-2,2-difluoro-myo-Ins-1-O-dodecylphosphonate were 8.3-fold and 4.8-fold less effective, respectively, than VP-616L. Methyl 2-deoxy-2,2-difluoro-myo-Ins-1-O-dodecylphosphonate was inactive. Also, a hundredfold less PI-PLC is required to cleave a glycosylphosphatidylinositol (GPI) than is needed to cleave PI. Implied in these observations are the following: (i) in powerful inhibitors an active site residue probably interacts with the equatorially oriented fluoro substituent; (ii) substrate recognition requires a negative charge on the phosphoryl at the Ins-1 position, and (iii) a GPI is better substrate than PI, for PI-PLC. Aminoglycoside antibiotics kanamycin A, gentamycin, and G418 stimulated PI-PLC cleavage of the GPI anchor of variant surface glycoprotein (VSG) from Trypanosoma brucei 2- to 4-fold. G418, which appears to act on the enzyme.substrate complex, increased kcat and Km 6.4-fold and 9.9-fold, respectively. PI-PLC was activated by G418 even in the presence of the inhibitor VP-616L. In control experiments, the lectin concanavalin A (ConA), which probably acts by substrate sequestration, inhibited both PI-PLC (Xi(50) = 0.00025) and GPI-specific phospholipase D (Xi(50) = 0.00018). G418 failed to activate PI-PLC when ConA was present. These observations indicate that G418 is an allosteric activator of Bacillus cereus PI-PLC. Since G418 stimulates a purified enzyme that is not involved in aminoglycoside metabolism, we propose that binding of aminoglycosides to cellular proteins could contribute to the development of the nephrotoxicity associated with the use of these aminoglycoside antibiotics.
Morris
J C
JC
Department of Cellular Biology, The University of Georgia, Athens, Georgia 30602, USA.
Ping-Sheng
L
L
Zhai
H X
HX
Shen
T Y
TY
Mensa-Wilmot
K
K
D000494:Allosteric Regulation; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001409:Bacillus cereus / Q000201:enzymology; D001667:Binding, Competitive; D002417:Cattle; D002478:Cells, Cultured; D003208:Concanavalin A / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005839:Gentamicins / Q000494:pharmacology*; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D006801:Humans; D007294:Inositol / Q000031:analogs & derivatives* / Q000494:pharmacology; D007700:Kinetics; D063065:Organophosphonates; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors
C100389:2-deoxy-2-fluoro-scyllo-inositol-1-O-dodecylphosphonate; D000900:Anti-Bacterial Agents; D004791:Enzyme Inhibitors; D005839:Gentamicins; D017261:Glycosylphosphatidylinositols; D063065:Organophosphonates; D003208:Concanavalin A; D007294:Inositol; C010680:antibiotic G 418; D010727:Phosphoric Diester Hydrolases; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; D043265:Phosphatidylinositol Diacylglycerol-Lyase