Analyses of co-operative transitions in Plasmodium falciparum beta-ketoacyl acyl carrier protein reductase upon co-factor and acyl carrier protein binding.
The type II fatty acid synthase pathway of Plasmodium falciparum is a validated unique target for developing novel antimalarials because of its intrinsic differences from the type I pathway operating in humans. beta-Ketoacyl-acyl carrier protein reductase is the only enzyme of this pathway that has no isoforms and thus selective inhibitors can be developed for this player of the pathway. We report here intensive studies on the direct interactions of Plasmodiumbeta-ketoacyl-acyl carrier protein reductase with its cofactor, NADPH, acyl carrier protein, acetoacetyl-coenzyme A and other ligands in solution, by monitoring the intrinsic fluorescence (lambdamax 334 nM) of the protein as a result of its lone tryptophan, as well as the fluorescence of NADPH (lambdamax 450 nM) upon binding to the enzyme. Binding of the reduced cofactor makes the enzyme catalytically efficient, as it increases the binding affinity of the substrate, acetoacetyl-coenzyme A, by 16-fold. The binding affinity of acyl carrier protein to the enzyme also increases by approximately threefold upon NADPH binding. Plasmodiumbeta-ketoacyl-acyl carrier protein reductase exhibits negative, homotropic co-operative binding for NADPH, which is enhanced in the presence of acyl carrier protein. Acyl carrier protein increases the accessibility of NADPH to beta-ketoacyl-acyl carrier protein reductase, as evident from the increase in the accessibility of the tryptophan of beta-ketoacyl-acyl carrier protein reductase to acrylamide, from 81 to 98%. In the presence of NADP+, the reaction proceeds in the reverse direction (Ka=23.17 microM-1). These findings provide impetus for exploring the influence of ligands on the structure-activity relationship of Plasmodiumbeta-ketoacyl-acyl carrier protein reductase.
Karmodiya
Krishanpal
K
Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore, India.
Surolia
Namita
N
D064431:3-Oxoacyl-(Acyl-Carrier-Protein) Reductase; D000213:Acyl Carrier Protein / Q000378:metabolism*; D000214:Acyl Coenzyme A / Q000378:metabolism*; D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D000494:Allosteric Regulation; D000818:Animals; D003001:Cloning, Molecular; D007700:Kinetics; D010963:Plasmodium falciparum / Q000201:enzymology*; D011485:Protein Binding
D000213:Acyl Carrier Protein; D000214:Acyl Coenzyme A; C010667:acetoacetyl CoA; D000429:Alcohol Oxidoreductases; D064431:3-Oxoacyl-(Acyl-Carrier-Protein) Reductase