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 an homodimeric metalloenzyme, containing one Mg2+ and two Zn2+ ions in each active site. ApoBIAP, prepared using ion-chelating agents, exhibited a dramatic decrease of its hydrolase activity, concomittant to conformational changes in its quaternary structure. By rate-zonal centrifugation and electrophoresis, we demonstrated, for the first time, that the loss of divalent ions leads to some monomerization process for a metal-depleted alkaline phosphatase. Divalent ions are also involved in the secondary and tertiary structures. Metal-depletion induced more exposure of some Trp residues and hydrophobic 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-sheets (as probed by FTIR spectroscopy). For BIAP, three steps of temperature-induced changes were exhibited, while for apoBIAP, only one step was exhibited at 55 degrees C. Our work on BIAP showed two main differences with alkaline phosphatase from Escherichia coli. The loss of the divalent ions induces protein monomerization and the total recovery of enzyme activity by divalent ion addition to apoBIAP was not obtained.
Bortolato
M
M
Laboratoire de Physico-Chimie Biologique, Université Claude Bernard Lyon I, Villeurbanne, France. bortolat@univ-lyon1.fr
Besson
F
F
Roux
B
B
D000469:Alkaline Phosphatase / Q000032:analysis / Q000737:chemistry*; D000818:Animals; D001051:Apoenzymes / Q000032:analysis / Q000737:chemistry; D002417:Cattle; D007413:Intestinal Mucosa / Q000737:chemistry*; D008274:Magnesium / Q000737:chemistry*; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D015032:Zinc / Q000737:chemistry*
D001051:Apoenzymes; D000469:Alkaline Phosphatase; D008274:Magnesium; D015032:Zinc