Ionic States of Substrates and Transition State Analogues at the Catalytic Sites ofN-Ribosyltransferases†

Anthony A. Sauve, Sean M. Cahill, Stephan G. Zech, Luiz A. Basso, Andrzej Lewandowicz, Diogenes S. Santos, Charles Grubmeyer, Gary B. Evans, Richard H. Furneaux, Peter C. Tyler, Ann McDermott, Mark E. Girvin (+1 others)
2003 Biochemistry  
Purine nucleoside phosphorylase (PNP) and hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) catalyze N-ribosidic bond cleavage in purine nucleosides and nucleotides, with addition of phosphate or pyrophosphate to form phosphorylated R-D-ribose products. The transition states have oxacarbenium ion character with a positive charge near 1′-C and ionic stabilization from nearby phosphoryl anions. Immucillin-H (ImmH) and Immucillin-H 5′-PO 4 (ImmHP) resemble the transition state charge when
more » ... state charge when protonated at 4′-N and bind tightly to these enzymes with K d values of 20 pM to 1 nM. It has been proposed that Immucillins bind as the 4′-N neutral form and are protonated in the slow-onset step. Solution and solid-state NMR spectra of ImmH, ImmHP, guanosine, and GMP in complexes with two PNPs and a HGPRTase have been used to characterize their ionization states. Results with PNP•ImmH•PO 4 and HGPRTase•ImmHP•MgPP i indicate protonation at N-4′ for the tightly bound inhibitors. The 1′-13 C and 1′-1 H resonances of bound Immucillins showed large downfield shifts as compared to Michaelis complexes, suggesting distortion of 1′-C toward sp 2 geometry. The Immucillins act as transition state mimics by binding with neutral iminoribitol groups followed by 4′-N protonation during slow-onset inhibition to form carbocationic mimics of the transition states. The ability of the Immucillins to mimic both substrate and transition state features contributes to their capture of transition state binding energy. Enzyme-activated phosphoryl nucleophiles bound to PNP and HGPRTase suggest enhanced electrostatic stabilization of the cationic transition states. Distortion of the oxacarbenium ion mimic toward transition state geometry is a common feature of the three distinct enzymatic complexes analyzed here. Substrate complexes, even in catalytically cycling equilibrium mixtures, do not reveal similar distortions. FIGURE 1: Chemical structures of ImmH and ImmH 5′-phosphate (upper panel). The lower panel indicates the catalytic site contacts for ImmH and phosphate at the catalytic site of M. tuberculosis PNP (A) and for ImmHP and MgPPi at the catalytic sites of P. falciparum HGPRTase (B) (15, 18).
doi:10.1021/bi034003a pmid:12741826 fatcat:znvlfzberfazzbvoa4tabxcm6q