Kinetic and structural analysis of the increased affinity of enoyl-ACP (acyl-carrier protein) reductase for triclosan in the presence of $NAD^{\mathrm{+}}$

Kapoor, Mili and Mukhi, Swarna PL and Surolia, Namita and Suguna, K and Surolia, Avadhesha (2004) Kinetic and structural analysis of the increased affinity of enoyl-ACP (acyl-carrier protein) reductase for triclosan in the presence of $NAD^{\mathrm{+}}$. In: Biochemical Journal, 381 . pp. 725-733.

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Abstract

The binding of enoyl-ACP (acyl-carrier protein) reductase from Plasmodium falciparum (PfENR) with its substrates and inhibitors has been analysed by SPR (surface plasmon resonance). The binding of the substrate analogue crotonoyl-CoA and coenzyme NADH to PfENR was monitored in real time by observing changes in response units. The binding constants determined for crotonoyl-CoA and NADH were 1.6 x $10^{\mathrm{4}}M^{\mathrm{-1}}$ and 1.9 x $10^{\mathrm{4}} M^{\mathrm{-1}}$ respectively. Triclosan,which has recently been demonstrated as a potent antimalarial agent, bound to the enzyme with a binding constant of 1.08 x $10^{\mathrm{5}} M^{\mathrm{-1}}$. However, there was a 300-fold increase in the binding constant in the presence of $NAD^{\mathrm{+}}$.The increase in the binding constant was due to a 17 times increase in the association rate constant $(k_{1})$ from $741 M^{\mathrm{-1}}$.$s^{\mathrm{-1}}$ to 1.3 x$10^{\mathrm{4}} M^{\mathrm{-1}}$.$s^{\mathrm{-1}}$ and a 16 times decrease in the dissociation rate constant $(k_{-1})$ from 6.84 x$10^{\mathrm{-3}} s^{\mathrm{-1}}$ to 4.2 x$10^{\mathrm{-4}} s^{\mathrm{-1}}$. These values are in agreement with those determined by steady-state kinetic analysis of the inhibition reaction [Kapoor, Reddy, Krishnasastry, N.Surolia and A. Surolia (2004) Biochem. J. 381, 719-724]. In SPR experiments, the binding of NAD(+) to PfENR was not detected. However,a binding constant of 6.5 x $10^{\mathrm{4}} M^{\mathrm{-1}}$ was obtained in the presence of triclosan. Further support for these observations was provided by the crystal structures of the binary and ternary complexes of PfENR. Thus the dramatic enhancement in the binding affinity of both triclosan and $NAD^{\mathrm{+}}$ in the ternary complex can be explained by increased van der Waals contacts in the ternary complex, facilitated by the movement of residues 318-324 of the substrate-binding loop and the nicotinamide ring of $NAD^{\mathrm{+}}$. Interestingly, the results of the present study also provide a rationale for the increased affinity of $NAD^{\mathrm{+}}$ for the enzyme in the ternary complex.

Item Type:	Journal Article
Publication:	Biochemical Journal
Publisher:	Portland Press
Additional Information:	Copyright for this article belongs to Portland Press.
Keywords:	antimalarial drug;enoyl-ACP (acyl-carrier protein) reductase;fatty acid biosynthesis;malaria;surface plasmon resonance;triclosan.
Department/Centre:	Division of Biological Sciences > Molecular Biophysics Unit
Date Deposited:	22 Jan 2007
Last Modified:	05 Jan 2012 10:26
URI:	http://eprints.iisc.ac.in/id/eprint/36

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