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

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• 作者：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.
• 刊名：Biochemistry
• 出版年：2003
• 出版时间：May 20, 2003
• 年：2003
• 卷：42
• 期：19
• 页码：5694 - 5705
• 全文大小：309K
• 年卷期：v.42,no.19(May 20, 2003)
• ISSN：1520-4995

文摘

Purine nucleoside phosphorylase (PNP) and hypoxanthine-guanine phosphoribosyltransferase(HGPRTase) catalyze N-ribosidic bond cleavage in purine nucleosides and nucleotides, with addition ofphosphate or pyrophosphate to form phosphorylated pha.gif" BORDER=0>-D-ribose products. The transition states haveoxacarbenium ion character with a positive charge near 1'-C and ionic stabilization from nearby phosphorylanions. Immucillin-H (ImmH) and Immucillin-H 5'-PO₄ (ImmHP) resemble the transition state chargewhen protonated at 4'-N and bind tightly to these enzymes with K_d 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 PNPImmHPO₄ andHGPRTaseImmHPMgPP_i indicate protonation at N-4' for the tightly bound inhibitors. The 1'-¹³C and1'-¹H resonances of bound Immucillins showed large downfield shifts as compared to Michaelis complexes,suggesting distortion of 1'-C toward sp² geometry. The Immucillins act as transition state mimics bybinding with neutral iminoribitol groups followed by 4'-N protonation during 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 binding energy. Enzyme-activatedphosphoryl nucleophiles bound to PNP and HGPRTase suggest 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 cycling equilibrium mixtures, do not reveal similar distortions.