Function assignment to conserved residues in mammalian alkaline phosphatases.

We have probed the structural/functional relationship of key residues in human placental alkaline phosphatase (PLAP) and compared their properties with those of the corresponding residues in Escherichia coli alkaline phosphatase (ECAP). Mutations were introduced in wild-type PLAP, i.e. [E429]PLAP, and in some instances also in [G429]PLAP, which displays properties characteristic of the human germ cell alkaline phosphatase isozyme. All active site metal ligands, as well as residues in their vicinity, were substituted to alanines or to the homologous residues present in ECAP. We found that mutations at Zn2 or Mg sites had similar effects in PLAP and ECAP but that the environment of the Zn1 ion in PLAP is less affected by substitutions than that in ECAP. Substitutions of the Mg and Zn1 neighboring residues His-317 and His-153 increased k(cat) and increased K(m) when compared with wild-type PLAP, contrary to what was predicted by the reciprocal substitutions in ECAP. All mammalian alkaline phosphatases (APs) have five cysteine residues (Cys-101, Cys-121, Cys-183, Cys-467, and Cys-474) per subunit, not homologous to any of the four cysteines in ECAP. By substituting each PLAP Cys by Ser, we found that disrupting the disulfide bond between Cys-121 and Cys-183 completely prevents the formation of the active enzyme, whereas the carboxyl-terminally located Cys-467-Cys-474 bond plays a lesser structural role. The substitution of the free Cys-101 did not significantly affect the properties of the enzyme. A distinguishing feature found in all mammalian APs, but not in ECAP, is the Tyr-367 residue involved in subunit contact and located close to the active site of the opposite subunit. We studied the A367 and F367 mutants of PLAP, as well as the corresponding double mutants containing G429. The mutations led to a 2-fold decrease in k(cat), a significant decrease in heat stability, and a significant disruption of inhibition by the uncompetitive inhibitors l-Phe and l-Leu. Our mutagenesis data, computer modeling, and docking predictions indicate that this residue contributes to the formation of the hydrophobic pocket that accommodates and stabilizes the side chain of the inhibitor during uncompetitive inhibition of mammalian APs.