MAMMALIAN ALKALINE-PHOSPHATASES ARE ALLOSTERIC ENZYMES

Mammalian alkaline phosphatases (APs) are zinc-containing metalloenzym es encoded by a multigene family and functional as dimeric molecules, Using human placental AP (FLAP) as a paradigm, we have investigated wh ether the monomers in a given FLAP dimer are subject to cooperativity during catalysis following an allosteric model or act via a half-of-si tes model, in which at any time only one single monomer is operative. Wild type and mutant FLAP homodimers and heterodimers were produced by stably transfecting Chinese hamster ovary cells with mutagenized FLAP cDNAs followed by enzyme extraction, purification, and characterizati on [Gly(429)]PLAP manifested negative cooperativity when partially met alated as a consequence of the reduced affinity of the incompletely me talated AP monomers for the substrate, Upon full metalation with Zn2+, however the negative cooperativity disappeared, To distinguish betwee n an allosteric and a half-of-sites model, a [Gly(429)]PLAP-[Ser(84)]P LAP heterodimer was produced by combining monomers displaying high and low sensitivity to the uncompetitive inhibitor L-Leu as well as a [Gl y(429)]PLAP-[Ala(92)]PLAP heterodimer combining a catalytically active and inactive monomer, respectively. The L-Leu inhibition profile of t he [Gly(429)]PLAP-[Ser(84)]PLAP heterodimer was intermediate to that f or each homodimer as predicted by the allosteric model. Likewise, the [Gly(429)]PLAP-[Ala(92)]PLAP heterodimer was catalytically active, con firming that AP monomers act independently of each other, Although het erodimers are structurally asymmetrical, they migrate in starch gels w ith a smaller than expected weighted electrophoretic mobility, are mor e 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 allost eric enzymes but that the stability and catalytic properties of each m onomer are controlled by the conformation of the second AP subunit.