Quantum mechanics/molecular mechanics investigation of the mechanism of phosphate transfer in human uridine-cytidine kinase 2

Abstract

The mechanisms of enzyme-catalyzed phosphate transfer and hydrolysis reactions are of great interest due to their importance and abundance in biochemistry. The reaction may proceed in a stepwise fashion, with either a pentavalent phosphorane or a metaphosphate anion intermediate, or by a concerted S N 2 mechanism. Despite much theoretical work focused on a few key enzymes, a consensus for the mechanism has not been reached, and examples of all three possibilities have been demonstrated. We have investigated the mechanism of human uridine-cytidine kinase 2 (UCK2, EC 2.7.1.48), which catalyzes the transfer of a phosphate group from ATP to the ribose 5′-hydroxyl of cytidine and uridine. UCK2 is normally expressed in human placenta, but is overexpressed in certain cancer cells, where it is responsible for activating a class of antitumor prodrugs. The UCK2 mechanism was investigated by generating a 2D potential energy surface as a function of the P-O bonds forming and breaking, with energies calculated using a quantum mechanics/molecular mechanics potential (B3LYP/6-31G(d):AMBER). The mechanism of phosphate transfer is shown to be concerted, and is accompanied by concerted proton transfer from the 5′-hydroxyl to a conserved active site aspartic acid that serves as a catalytic base. The calculated barrier for this reaction is 15.1 kcal/mol, in relatively good agreement with the experimental barrier of 17.5 kcal/mol. The interactions of the enzyme active site with the reactant, transition state, and product are examined for their implications on the design of anticancer prodrugs or positron emission tomography (PET) reporter probes for this enzyme.

Crude Extracts, Flavokawain B and Alpinetin Compounds from the Rhizome of Alpinia mutica Induce Cell Death via UCK2 Enzyme Inhibition and in Turn Reduce 18S rRNA Biosynthesis in HT-29 Cells

“…Overexpression of this enzyme have been implicated in several cancers and it is therefore considered a hallmark of cancer. The selective expression and non-immunogenicity of human UCK2 may, however represent a potential target for anticancer drug development [2]. …”

Structures of SAICAR synthetase (PurC) from Streptococcus pneumoniae with ADP, Mg2+, AIR and Asp

“…It is most likely that the activation of the carboxylate of CAIR is a relay process initiated by phosphate transfer to the carboxylate of a conserved side chain (1 in Fig. 5b; Smith et al, 2009). For ATP in the bent conformation, the carboxylate group of the Glu178 side chain is within van der Waals distance of the -phosphate ( Fig.…”

Structural and Biochemical Studies on the Reaction Mechanism of Uridine–Cytidine Kinase

“…A lack of knowledge concerning the structure of the ternary complex with both substrates present hinders a complete understanding of the molecular mechanism of the phosphate transfer from ATP to nucleoside. Although a model of the initial stage of the UCK catalyzed reaction has been proposed [11,13], there has been no experimental evidence to support this mechanism.…”