Ionic states of substrates and transition state analogues at the catalytic sites of N-ribosyltransferases.
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 alpha-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 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.
Sauve
Anthony A
AA
Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
Cahill
Sean M
SM
Zech
Stephan G
SG
Basso
Luiz A
LA
Lewandowicz
Andrzej
A
Santos
Diogenes S
DS
Grubmeyer
Charles
C
Evans
Gary B
GB
Furneaux
Richard H
RH
Tyler
Peter C
PC
McDermott
Ann
A
Girvin
Mark E
ME
Schramm
Vern L
VL
D020134:Catalytic Domain; D006151:Guanosine / Q000737:chemistry / Q000378:metabolism; D006157:Guanosine Monophosphate / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007041:Hypoxanthine Phosphoribosyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D066298:In Vitro Techniques; D007477:Ions / Q000737:chemistry; D007700:Kinetics; D015394:Molecular Structure; D009169:Mycobacterium tuberculosis / Q000201:enzymology / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D010766:Phosphorylation; D011684:Purine Nucleosides; D011683:Purine-Nucleoside Phosphorylase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011744:Pyrimidinones / Q000737:chemistry / Q000378:metabolism; D011758:Pyrroles / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D007477:Ions; D011684:Purine Nucleosides; D011744:Pyrimidinones; D011758:Pyrroles; D011994:Recombinant Proteins; D006151:Guanosine; C113101:forodesine; D006157:Guanosine Monophosphate; D011683:Purine-Nucleoside Phosphorylase; D007041:Hypoxanthine Phosphoribosyltransferase