Abstract
In enzymatic depurination of nucleosides, the 5'-OH group of the ribose moiety of the substrate is often shown to contribute substantially to catalysis. The purine-specific nucleoside hydrolase from Trypanosoma vivax (TvNH) fixes the 5'-OH group in a gauche,trans orientation about the C4'-C5' bond, enabling the 5'-oxygen to accept an intramolecular hydrogen bond from the C8-atom of the purine leaving group. High level ab initio quantum chemical calculations indicate that this interaction promotes protonation of the purine at N7. Steady state kinetics comprising engineered substrates confirm that a considerable fraction of the catalytic 5'-OH effect can be attributed to leaving group activation.
Highlights
The 5Ј-OH Group in TvNH Does Not Contribute to Ribooxocarbenium Ion Stabilization—In all NHs, the 5Ј-OH group of the ribose moiety of the substrate has been shown to play a prominent role in catalysis [1,2,3,4]
In the NHs [10], as well as in purine nucleoside phosphorylase [19], this 5Ј-OH catalytic effect has been proposed to originate from a stabilization of the ribo-oxocarbenium transition state via an electrostatic neighboring group interaction with the O4Ј atom
This proposal was based on crystal structures of the enzymes in complex with substrate analogues or transition state inhibitors, invariably showing the 5Ј-OH group firmly anchored in a gauche,trans orientation about the C4Ј–C5Ј bond
Summary
A similar anchoring of the 5Ј-OH is observed in the homologous and extensively studied C. fasciculata NH [10] In the latter enzyme as well, the 5Ј-OH was found to be crucial for catalysis [2], and it was suggested that the lone pair electrons of the 5Ј-oxygen stabilizes the ribo-oxocarbenium transition state electrostatically via a “neighboring group interaction” with O4Ј (Fig. 1a, dashed line) [5, 10]. Such an interaction would imply that the oxonium-like resonance form of the oxocarbenium predominates (Fig. 1b). We set out to investigate the catalytic role of the 5Ј-OH by theoretical and experimental methods
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