Role of metal ions on the secondary and quaternary structure of alkaline phosphatase from bovine intestinal mucosa

Alkaline phosphatase (EC 3.1.3,1) from bovine intestinal mucosa (BIAP) is a n homodimeric metalloenzyme, containing one Mg2+ and two Zn2+ ions in each active site. ApoBIAP, prepared using ion-chelating agents, exhibited a dram atic decrease of its hydrolase activity, concomittant to conformational cha nges in its quaternary structure, By rate-zonal centrifugation and electrop horesis, we demonstrated, for the first time, that the loss of divalent ion s leads to some monomerization process for a metal-depleted alkaline phosph atase, Divalent ions art! also involved in the secondary and tertiary struc tures, Metal-depletion induced more exposure of some Trp residues and hydro phobic regions to the solvent (as proved by intrinsic and ANS fluorescences ), These changes might correspond to the disappearance of alpha-helices and /or turns with a concomittant appearance of unordered structures and beta-s heets (as probed by FTIR, spectroscopy). For BIAP, three steps of temperatu re-induced changes were exhibited, while for apoBIAP, only one step was exh ibited at 55 degrees C, Our work on BIAP showed two main differences with a lkaline phosphatase from Escherichia coli, The loss of the divalent ions in duces protein monomerization and the total recovery of enzyme activity by d ivalent; ion addition to apoBIAP was not obtained, Proteins 1999;37:310-318 , (C) 1999 Wiley-Liss,Inc.