Plasmodium falciparum hypoxanthine guanine phosphoribosyltransferase. Stability studies on the product-activated enzyme.
Hypoxanthine guanine phosphoribosyltransferases (HGPRTs) catalyze the conversion of 6-oxopurine bases to their respective nucleotides, the phosphoribosyl group being derived from phosphoribosyl pyrophosphate. Recombinant Plasmodium falciparum HGPRT, on purification, has negligible activity, and previous reports have shown that high activities can be achieved upon incubation of recombinant enzyme with the substrates hypoxanthine and phosphoribosyl pyrophosphate [Keough DT, Ng AL, Winzor DJ, Emmerson BT & de Jersey J (1999) Mol Biochem Parasitol98, 29-41; Sujay Subbayya IN & Balaram H (2000) Biochem Biophys Res Commun279, 433-437]. In this report, we show that activation is effected by the product, Inosine monophosphate (IMP), and not by the substrates. Studies carried out on Plasmodium falciparum HGPRT and on a temperature-sensitive mutant, L44F, show that the enzymes are destabilized in the presence of the substrates and the product, IMP. These stability studies suggest that the active, product-bound form of the enzyme is less stable than the ligand-free, unactivated enzyme. Equilibrium isothermal-unfolding studies indicate that the active form is destabilized by 2-3 kcal x mol(-1) compared with the unactivated state. This presents a unique example of an enzyme that attains its active conformation of lower stability by product binding. This property of ligand-mediated activation is not seen with recombinant human HGPRT, which is highly active in the unliganded state. The reversibility between highly active and weakly active states suggests a novel mechanism for the regulation of enzyme activity in P. falciparum.
Raman
Jayalakshmi
J
Molecular Biology and Genetics Unit, Jawarharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore, India.
Ashok
Chethan S
CS
Subbayya
Sujay I N
SI
Anand
Ranjith P
RP
Selvi
Senthamizh T
ST
Balaram
Hemalatha
H
D000818:Animals; D002942:Circular Dichroism; D004789:Enzyme Activation; D004795:Enzyme Stability; D019791:Guanidine / Q000494:pharmacology; D006801:Humans; D019271:Hypoxanthine / Q000737:chemistry / Q000378:metabolism; D007041:Hypoxanthine Phosphoribosyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007291:Inosine Monophosphate / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D013696:Temperature; D013816:Thermodynamics
D007291:Inosine Monophosphate; D019271:Hypoxanthine; D007041:Hypoxanthine Phosphoribosyltransferase; D019791:Guanidine