Escherichia coli 5′‐nucleotidase (5′‐NT) is a monomeric protein of 525 amino acids (58.1 kDa). The enzyme has a nutritional function in bacteria where it is exported to the periplasmic space to hydrolyze external nucleotides and UDP‐glucose. Related animal 5′‐nucleotidases are attached to the cell membrane via a GPI anchor and hydrolyze nucleotides as extracellular signaling substances. E. coli 5′‐NT belongs to the calcineurin superfamily of metallophosphatases and contains two divalent metal ions at a distance of about 3.3 Å. One of these metal ions is probably Zn 2+ in vivo . However, the Zn 2+ –Zn 2+ form is relatively inactive, whereas good activity is seen in the presence of Mg 2+ , Mn 2+ , and Co 2+ . The protein consists of two domains: a larger N‐terminal domain, which is related to the calcineurin superfamily and contains the two catalytic metal ions and its ligands, and the C‐terminal domain, which bears the substrate specificity pocket that binds the nucleoside moiety of the substrates. Thus, the active site is located at the interface between both domains. In the proposed catalytic mechanism, both metal ions are involved in substrate binding, activation of the nucleophile, and transition‐state stabilization. In addition, a catalytic histidine residue (His117 in E. coli 5′‐NT) is strictly conserved in all enzymes of this superfamily.

Leucine aminopeptidase belongs to the metallohydrolase family M17. In this family, two X‐ray diffraction structures are known for bovine lens LAP (blLAP) and for the Escherichia coli aminopeptidase A (PepA). These two peptidases are large homohexamers of 32 symmetry, and both have a large central solvent cavity near the active sites. Each active center contains two Zn 2+ separated by 3.0 Å. In PepA, maximal activity occurs in the presence of Mn 2+ . blLAP is activated by Mn 2+ , Mg 2+ or Co 2+ , but zinc is bound most strongly and is supposed to be the physiological cofactor. In the proposed catalytic mechanism, both metal ions stabilize substrate binding and formation of the transition state. In addition, a lysine side chain contributes by polarizing the substrate's carbonyl group, and an arginine side chain binds a bicarbonate anion (or carbonate ion) that is in a position to act, at least in PepA, as a general base to deprotonate the zinc‐bound water nucleophile.