Elucidation of the substrate specificity, kinetic and catalytic mechanism of adenylosuccinate lyase from Plasmodium falciparum.
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.
Bulusu
Vinay
V
Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore 560 064, India.
Srinivasan
Bharath
B
Bopanna
Monnanda Ponnappa
MP
Balaram
Hemalatha
H
D000249:Adenosine Monophosphate / Q000378:metabolism; D000264:Adenylosuccinate Lyase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000620:Aminoimidazole Carboxamide / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D055162:Biocatalysis; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012265:Ribonucleotides / Q000378:metabolism / Q000494:pharmacology; D016415:Sequence Alignment; D013379:Substrate Specificity; D013816:Thermodynamics
D015800:Protozoan Proteins; D011994:Recombinant Proteins; D012265:Ribonucleotides; D000620:Aminoimidazole Carboxamide; C021330:succinyladenosine 3',5'-cyclic monophosphate; D000249:Adenosine Monophosphate; D000242:Cyclic AMP; D000264:Adenylosuccinate Lyase; C031143:AICA ribonucleotide; C020635:SAICAR