A unique and differential effect of denaturants on cofactor mediated activation of Plasmodium falciparum beta-ketoacyl-ACP reductase.
The urea and guanidinium chloride (GdmCl) induced unfolding of FabG, a beta-ketoacyl-ACP reductase of Plasmodium falciparum, was examined in detail using intrinsic fluorescence of FabG, UV-circular dichroism (CD), spectrophotometric enzyme activity measurements, glutaraldehyde cross-linking, and size exclusion chromatography. The equilibrium unfolding of FabG by urea is a multistep process as compared with a two-state process by GdmCl. FabG is fully unfolded at 6.0M urea and 4.0M GdmCl. Approximately 90% of the enzyme activity could be recovered on dialyzing the denaturants, showing that denaturation by both urea and GdmCl is reversible. We found two states in the reversible unfolding process of FabG in presence of NADPH; one is an activity-enhanced state and the other, an inactive state in case of equilibrium unfolding with urea. On the contrary, in presence of NADPH, there is no stabilization of FabG in case of equilibrium unfolding with GdmCl. We hypothesize that the hydrogen-bonding network may be reorganized by the denaturant in the activity-enhanced state formed in presence of 1.0M urea, by interrupting the association between dimer-dimer interface and help in accommodating the larger substrate in the substrate binding tunnel thus, increasing the activity. Furthermore, binding of the active site organizer, NADPH leads to compaction of the FabG in presence of urea, as evident by acrylamide quenching. We have shown here for the first time, the detailed inactivation kinetics of FabG, which have not been evaluated in the past from any of the FabG family of enzymes from any of the other sources. These findings provide impetus for exploring the influences of ligands on the structure-activity relationship of Plasmodium beta-ketoacyl-ACP reductase.
Karmodiya
Krishanpal
K
Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore 560064, India.
Surolia
Namita
N
D064431:3-Oxoacyl-(Acyl-Carrier-Protein) Reductase; D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004789:Enzyme Activation; D019791:Guanidine / Q000737:chemistry; D010963:Plasmodium falciparum / Q000201:enzymology*; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D014508:Urea / Q000737:chemistry
D014508:Urea; D000429:Alcohol Oxidoreductases; D064431:3-Oxoacyl-(Acyl-Carrier-Protein) Reductase; D019791:Guanidine