Purine nucleoside phosphorylase (PNP) and hypoxanthine-
guanine phosphoribosyltransferase(HGPRTase) catalyze N-ribosidic bond cleava
ge in purine nucleosides and nucleotides, with addition ofphosphate or pyrophosphate to form phosphorylated
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D-ribose products. The transition states haveoxacarbenium ion character with a positive char
ge near 1'-C and ionic stabilization from nearby phosphorylanions. Immucillin-H (ImmH) and Immucillin-H 5'-PO
4 (ImmHP) resemble the transition state char
gewhen protonated at 4'-N and bind ti
ghtly to these enzymes with
Kd values of 20 pM to 1 nM. It has beenproposed that Immucillins bind as the 4'-N neutral form and are protonated in the slow-onset step. Solutionand solid-state NMR spectra of ImmH, ImmHP,
guanosine, and GMP in complexes with two PNPs anda HGPRTase have been used to characterize their ionization states. Results with PNP
ges/entities/bull.
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ges/entities/bull.
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4 andHGPRTase
ges/entities/bull.
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ges/entities/bull.
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gPP
i indicate protonation at N-4' for the ti
ghtly bound inhibitors. The 1'-
13C and1'-
1H resonances of bound Immucillins showed lar
ge downfield shifts as compared to Michaelis complexes,su
ggestin
g distortion of 1'-C toward sp
2 geometry. The Immucillins act as transition state mimics bybindin
g with neutral iminoribitol
groups followed by 4'-N protonation durin
g slow-onset inhibition toform carbocationic mimics of the transition states. The ability of the Immucillins to mimic both substrateand transition state features contributes to their capture of transition state bindin
g ener
gy. Enzyme-activatedphosphoryl nucleophiles bound to PNP and HGPRTase su
ggest enhanced electrostatic stabilization of thecationic transition states. Distortion of the oxacarbenium ion mimic toward transition state
geometry is acommon feature of the three distinct enzymatic complexes analyzed here. Substrate complexes, even incatalytically cyclin
g equilibrium mixtures, do not reveal similar distortions.