Enzyme‐catalyzed uridine phosphorolysis: S_N2 mechanism with phosphate activation by desolvation

Abstract

The rate of uridine phosphorolysis catalyzed by uridine phosphorylase from Escherichia coli decreases with increasing ionic strength. In contrast, the rate was increased about twofold after preincubation of uridine phosphorylase with 60% acetonitrile. These data correlate with known effects of polar and bipolar aprotic solvents on SN2 nucleophilic substitution reactions. The enzyme modified with fluorescein-5'-isothiocyanate (fluorescein residue occupies an uridine-binding subsite [Komissarov et al., (1994) Biochim. Biophys. Acta 1205, 54-58]) was selectively modified with irreversible inhibitor SA-423, which reacts near the phosphate-binding subsite. The double-modified uridine phosphorylase is assumed to imitate the enzyme-substrate complex. Modification with SA-423 was accompanied with dramatic changes in the absorption spectrum of active site-linked fluorescein, which were identical to those for fluorescein in a hydrophobic medium, namely 80% acetonitrile. The data obtained suggest that an increase in active site hydrophobicity leads to phosphate desolvation and facilitates the enzymatic SN2 uridine phosphorolysis reaction.

Transition-State Analysis of Trypanosoma cruzi Uridine Phosphorylase-Catalyzed Arsenolysis of Uridine

“…S N 2 mechanisms have also been reported for E. coli UP phosphorolysis and for human TP-catalyzed arsenolysis of thymidine, however in the latter, bond breaking was significantly more advanced than bond making at the transition state …”

“…This is in contrast with the dissociative transition state models, with no significant participation of the attacking nucleophile, reported for inosine arsenolysis by bovine, malarial, and human PNP, 12,47 and for the hydrolytic cleavage of the N -glycosidic bond of 2′-deoxyuridine in DNA, catalyzed by E. coli uracil DNA glycosylase (UDG), 49 while in agreement with the almost synchronous, though less tight, transition state structure proposed for the NAD + hydrolysis catalyzed by diphtheria toxin. 44 S N 2 mechanisms have also been reported for E. coli UP phosphorolysis, 50 and for human TP-catalyzed arsenolysis of thymidine, however in the latter, bond breaking was significantly more advanced than bond making at the transition state. 16 …”

UDP-glucose Dehydrogenase: The First-step Oxidation Is an NAD+-dependent Bimolecular Nucleophilic Substitution Reaction (SN2)

“…To the best of our knowledge, this NAD + -dependent S N 2 mechanism has not been reported before, although many other enzymes are known to involve an S N 2 reaction, e.g. , haloalkane dehalogenase 42-45, uridine diphosphate glucuronosyltransferase 46, sorbitol dehydrogenase 47, and uridine phosphorylase 48-50. These S N 2 reactions are however independent of the cofactor NAD + .…”

Uridine Phosphorylase from Trypanosoma cruzi: Kinetic and Chemical Mechanisms

“…The chemical mechanism for UP reactions is formally an S N 2 mechanism, since the reaction catalyzes the inversion of stereochemistry at the ribosyl anomeric carbon. This mechanism has been suggested for E. coli UP . However, it is now well-known that enzymatic reactions with inversion of stereochemistry can be characterized by fully dissociated S N 1 transition states, and these types of transition states are established in N -ribosyltransferases, including bovine, human, and malarial purine nucleoside phosphorylases (PNPs) and human methylthioadenosine phosphorylase, which are also members of the NP-I family …”