Transition state inhibitors of 6-oxopurine phosphoribosyltransferases
Li, Caroline Ming-Way
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The protozoan parasite Plasmodium falciparum causes the most deadly form of malaria in humans. Resistance to current chemotherapeutic agents support the need for discovering new drugs. A promising antimalarial target is P. falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT), a key enzyme in salvaging purines needed to make nucleotides. Humans have an equivalent enzyme (HGPRT) that uses hypoxanthine or guanine as substrate. HG(X)PRT catalyzes transfer of the phosphoribosyl portion of alpha-D-5-phosphoribosyl 1-pyrophosphate to a 6-oxopurine forming the corresponding nucleotide and inorganic pyrophosphate.;Results from kinetic isotope effect experiments for P. falciparum HGXPRT support the mechanism where the bond breaking step in catalysis fast, Michaelis complex dissociates slowly to free substrate, and enzyme-product converts readily to enzyme-substrate. Even though the kinetic isotope effect results did not give enough information to solve the transition state structure of HGXPRT, the transition state structure was proposed based on kinetic isotope effect experiments of other 6-oxopurine phosphoribosyltransferases.;The inhibitors immucillinHP and immucillinGP were synthesized to mimic properties of the proposed transition state structure including protonated N7 and positive charge on the ribose. Inhibitors with proposed transition state features were characterized with nM inhibition constants for P. falciparum (malaria), S. mansoni (schistosomiasis), T. foetus (trichomoniasis), G. lamblia (giardiasis), and human 6-oxopurine phosphoribosyltransferase. Binding of these inhibitors is >1,000-fold tighter than other inhibitors reported for 6-oxopurine phosphoribosyltransferases. 1H NMR methods revealed enzyme conformational changes with substrate binding and identified a specific contact of the inhibitor to the enzyme involved in acid/base catalysis. P. falciparum and human HG(X)PRT complexed with inhibitor was crystallized for X-ray crystallography studies.;Evidence that these inhibitors are transition state inhibitors include: (1) slow-onset/tight binding inhibition from kinetic analysis, (2) interactions with N7 from NMR and X-ray crystallography studies, (3) oxocarbenium stabilization from X-ray crystallography analysis, and (4) ribosyl ring pucker from X-ray crystallography analysis. The inhibitors investigated in these studies are an important advancement in developing antimalarial and antiparasitic agents.
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