Elucidation of the substrate specificity, kinetic and catalytic mechanism of adenylosuccinate lyase from Plasmodium falciparum

Bulusu, Vinay ; Srinivasan, Bharath ; Bopanna, Monnanda Ponnappa ; Balaram, Hemalatha (2009) Elucidation of the substrate specificity, kinetic and catalytic mechanism of adenylosuccinate lyase from Plasmodium falciparum Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics, 1794 (4). pp. 642-654. ISSN 1570-9639

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Official URL: http://linkinghub.elsevier.com/retrieve/pii/S15709...

Related URL: http://dx.doi.org/10.1016/j.bbapap.2008.11.021

Abstract

Adenylosuccinate lyase (ASL) catalyzes two distinct but chemically similar reactions in purine biosynthesis. The first, exclusive to the de novo pathway involves the cleavage of 5-aminoimidazole-4-(N-succinylcarboxamide) ribonucleotide (SAICAR) to 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) and fumarate and the second common to both de novo and the salvage pathways involves the cleavage of succinyl-adenosine monophosphate (SAMP) to AMP and fumarate. A detailed kinetic and catalytic mechanism of the recombinant His-tagged ASL from Plasmodium falciparum (PfASL) is presented here. Initial velocity kinetics, product inhibition studies and transient kinetics indicate a Uni-Bi rapid equilibrium ordered mechanism. Substrate and solvent isotope effect studies implicate the process of C(γ)-N bond cleavage to be rate limiting. Interestingly, the effect of pH on kcat and kcat/Km highlight ionization of the base only in the enzyme substrate complex and not in the enzyme alone, thereby implicating the pivotal role of the substrate in the activation of the catalytic base. Site-directed mutagenesis implicates a key role for the conserved serine (S298) in catalysis. Despite the absence of a de novo pathway for purine synthesis and most importantly, the absence of other enzymes that can metabolise AICAR in P. falciparum, PfASL catalyzes the SAICAR cleavage reaction with kinetic parameters similar to those of SAMP reaction and binds AICAR with affinity similar to that of AMP. The presence of this catalytic feature allows the use of AICAR or its analogues as inhibitors of PfASL and hence, as novel putative anti-parasitic agents. In support of this, we do see a dose dependent inhibition of parasite growth in the presence of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAriboside) with half-maximal inhibition at 167 +/- 5 μ M.

Item Type:Article
Source:Copyright of this article belongs to Elsevier Science.
Keywords:Plasmodium Falciparum; Adenylosuccinate Lyase; Catalysis; Rapid Equilibrium; AICAR
ID Code:1255
Deposited On:04 Oct 2010 08:00
Last Modified:07 Jan 2011 10:22

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