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

TitleElucidation of the substrate specificity, kinetic and catalytic mechanism of adenylosuccinate lyase from Plasmodium falciparum
Publication TypeJournal Article
Year of Publication2009
AuthorsBulusu, V, Srinivasan B, Bopanna M P, Balaram H
JournalBiochimica et biophysica acta
Volume1794
Pagination642-54
Date Published2009 Apr
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(gamma)-N bond cleavage to be rate limiting. Interestingly, the effect of pH on k(cat) and k(cat)/K(m) 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 microM.

PDFhttp://cssb.biology.gatech.edu/skolnick/img/pdfs/Elucidation_Adenylosuccinate.pdf
Pub Med Link

http://www.ncbi.nlm.nih.gov/pubmed/19111634?dopt=Abstract

Alternate JournalBiochim. Biophys. Acta
Citekey19111634