Inhibition of glyceraldehyde-3-phosphate dehydrogenase by pentalenolactone: kinetic and mechanistic studies.
Incubation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with the antibiotic pentalenolactone (1) resulted in time-dependent, irreversible inhibition of GAPDH. The kinetics of inactivation were biphasic, exhibiting an initial rapid phase and a slower second phase. Pentalenolactone methyl ester (2) also irreversibly inactivated GADPH, albeit at a slower rate and with a higher KI. The substrate glyceraldehyde-3-phosphate (G-3-P) afforded protection against inactivation by 1, whereas the presence of NAD+ in the incubation mixture stimulated the inactivation by increasing the apparent affinity of the enzyme for the inhibitor. In steady-state kinetic experiments, 1 acted as a competitive inhibitor of GAPDH with respect to G-3-P but exhibited uncompetitive inhibition with respect to NAD+. Inactivation of NAD+-free apo-GAPDH by 1 showed simple pseudo-first-order kinetics. By titrating the free thiol residues of partially inactivated GAPDH, it was found that both pentalenolactone and pentalenolactone methyl ester react with all four Cys-SH residues of the tetrameric GAPDH.
Cane
D E
DE
Department of Chemistry, Brown University, Providence, Rhode Island 02912.
Sohng
J K
JK
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001667:Binding, Competitive; D002850:Chromatography, Gel; D005986:Glyceraldehyde 3-Phosphate / Q000378:metabolism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000037:antagonists & inhibitors*; D007700:Kinetics; D009132:Muscles / Q000201:enzymology*; D009243:NAD / Q000494:pharmacology; D011817:Rabbits; D012717:Sesquiterpenes / Q000494:pharmacology; D013223:Statistics as Topic; D013439:Sulfhydryl Reagents
D000900:Anti-Bacterial Agents; D012717:Sesquiterpenes; D013439:Sulfhydryl Reagents; D009243:NAD; D005986:Glyceraldehyde 3-Phosphate; C017996:arenaemycin E; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases