Exploring the active site of Trypanosoma brucei phosphofructokinase by inhibition studies: specific irreversible inhibition.
This work deals with the phosphofructokinase enzyme (PFK) of the parasite Trypanosoma brucei. Inhibitors which are analogues of fructose-6-phosphate (F6P) derived from 2,5-anhydromannitol and therefore blocked in a closed conformation, both nonphosphorylated and phosphorylated, were designed. They provided information on this class of ATP-dependent PFK (structurally more similar to PPi-dependent PFKs revealing (i) an ordered mechanism, ATP binding first, inducing an essential conformational change to increase the affinity for F6P, and (ii) a rather hydrophobic environment at the ATP binding site. Nonphosphorylated mannitol derivatives bind at both the ATP and F6P binding sites, whereas the phosphorylated derivatives only bind at the ATP binding site. The inhibitors bearing an aromatic ring substituted at the meta position indicate a polar interaction with lysine 227, which is specific to T. brucei PFK and is replaced by a glycine in human PFK. This lysine can be irreversibly bound, leading to inhibition when an electrophilic carbon atom is beta to the meta position on the ring. This lysine was identified by site-directed mutagenesis. This first example of a specific irreversible inactivation of T. brucei PFK offers an opportunity to develop biologically active compounds against the sleeping sickness, the causative agent of which is the trypanosome.
Claustre
Samantha
S
Laboratoire Synthèse et Physico Chimie de Molécules d'Intérêt Biologique UMR-CNRS 5068, Universitè Paul Sabatier, 118 route de Narbonne, 31062 Toulouse Cedex, France.
Denier
Colette
C
Lakhdar-Ghazal
Faouzi
F
Lougare
Andrée
A
Lopez
Claudia
C
Chevalier
Nathalie
N
Michels
Paul A M
PA
Périé
Jacques
J
Willson
Michèle
M
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002942:Circular Dichroism; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000494:pharmacology; D005636:Fructosephosphates / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D010732:Phosphofructokinase-1 / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods; D013379:Substrate Specificity; D014346:Trypanosoma brucei brucei / Q000201:enzymology*
D004791:Enzyme Inhibitors; D005636:Fructosephosphates; D011994:Recombinant Proteins; C027618:fructose-6-phosphate; D000255:Adenosine Triphosphate; D010732:Phosphofructokinase-1; D008274:Magnesium