Hydrolysis of the extracellular signalling substance ATP by 5'-nucleotidase

Biological background

5'-Nucleotidase (5'-NT) from E. coli is a zinc-containing enzyme, which is also known as UDP-sugar hydrolase. The periplasmic enzyme is probably involved in the hydrolysis of external UDP-glucose and of nucleotides, for utilization by the cell. Animal ecto-5'-nucleotidases are related to this bacterial enzyme. Ecto-nucleotidases are important for the formation and hydrolysis of nucleosides and nucleotides as extracellular signaling substances. Animal 5'-NTs function together with other ecto-nucleotidases in the termination of the neurotransmitter action of ATP in the brain and periphery.

Protein structure

We have crystallised the enzyme and determined the protein structure with the help of the MAD and MIR methods. The best crystals diffracted to 1.7 Angstrom resolution. The enzyme consists of a larger N-terminal domain (green) and a smaller C-terminal domain (blue). The two domains are linked by a long alpha-helix. Shown here is the structure of the closed (active) conformation. The catalytic center, which contains two zinc ions, is located at the interface between the two domains.

Cocrystal structures with inhibitors and products define the substrate specificity pocket and provide important information to understand the enzyme function. The inhibitor shown to the left (yellow carbon atoms) is a non-hydrolyzable analog of ADP, because the scissile anhydride bond between the phosphate groups is replaced by a methylene group. The enzyme was cocrystallized with the inhibitor, which indeed did bind to the catalytic site. Shown in green is the electron density map, obtained from the X-ray experiment, which defines the binding mode of the inhibitor.