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Mammalian Alkaline Phosphatases Are Allosteric Enzymes

1997; Elsevier BV; Volume: 272; Issue: 36 Linguagem: Inglês

10.1074/jbc.272.36.22781

1083-351X

Marc Hoylaerts, Thomas D. Manes, José Luís Millán,

Selenium in Biological Systems

Mammalian alkaline phosphatases (APs) are zinc-containing metalloenzymes encoded by a multigene family and functional as dimeric molecules. Using human placental AP (PLAP) as a paradigm, we have investigated whether the monomers in a given PLAP dimer are subject to cooperativity during catalysis following an allosteric model or act via a half-of-sites model, in which at any time only one single monomer is operative. Wild type and mutant PLAP homodimers and heterodimers were produced by stably transfecting Chinese hamster ovary cells with mutagenized PLAP cDNAs followed by enzyme extraction, purification, and characterization. [Gly 429 ]PLAP manifested negative cooperativity when partially metalated as a consequence of the reduced affinity of the incompletely metalated AP monomers for the substrate. Upon full metalation with Zn 2+ , however, the negative cooperativity disappeared. To distinguish between an allosteric and a half-of-sites model, a [Gly 429 ]PLAP-[Ser 84 ]PLAP heterodimer was produced by combining monomers displaying high and low sensitivity to the uncompetitive inhibitor l-Leu as well as a [Gly 429 ]PLAP-[Ala 92 ]PLAP heterodimer combining a catalytically active and inactive monomer, respectively. The l-Leu inhibition profile of the [Gly 429 ]PLAP-[Ser 84 ]PLAP heterodimer was intermediate to that for each homodimer as predicted by the allosteric model. Likewise, the [Gly 429 ]PLAP-[Ala 92 ]PLAP heterodimer was catalytically active, confirming that AP monomers act independently of each other. Although heterodimers are structurally asymmetrical, they migrate in starch gels with a smaller than expected weighted electrophoretic mobility, are more stable to heat denaturation than expected, and are more sensitive to l-Leu inhibition than predicted by a strict noncooperative model. We conclude that fully metalated mammalian APs are noncooperative allosteric enzymes but that the stability and catalytic properties of each monomer are controlled by the conformation of the second AP subunit.