Distinct structure and activity recoveries reveal differences in metal binding between mammalian and Escherichia coli alkaline phosphatases.
The amino acids involved in the coordination of two Zn2+ ions and one Mg2+ ion in the active site are well conserved from EAP (Escherichia coli alkaline phosphatase) to BIAP (bovine intestinal alkaline phosphatase), whereas most of their surrounding residues are different. To verify the consequences of this heterology on their specific activities, we compared the activity and structure recoveries of the metal-free forms (apo) of EAP and of BIAP. In the present study, we found that although the sensitivities of EAP and BIAP to ions remained similar, significant differences in dimeric structure stability of apo-enzymes were observed between EAP and BIAP, as well as in the kinetics of their activity and secondary structure recoveries. After mild chelation inactive apo-EAP was monomeric under mild denaturing conditions, whereas inactive apo-BIAP remained dimeric, indicating that the monomer-monomer contact was stronger in the mammalian enzyme. Dimeric apo-EAP (0.45 microM, corresponding to 4 units/ml) recovered approx. 80% of its initial activity after 3 min incubation in an optimal recovery medium containing 5 microM Zn2+ and 5 mM Mg2+, whereas dimeric apo-BIAP (0.016 microM, corresponding to 4 units/ml) recovered 80% of its native activity after 6 h incubation in an optimal recovery medium containing 0.5 microM Zn2+ and 5 mM Mg2+. Small and different secondary structure changes were also observed during activity recoveries of apo-BIAP and apo-EAP, which were not in parallel with the activity recoveries, suggesting that distinct and subtle structural changes are required for their optimal activity recoveries.
Zhang
Le
L
UFR de Chimie-Biochimie, CNRS-UMR 5013, Université Claude Bernard Lyon 1, 43 Boulevard du 11 Novembre 1918, 69622 Villeurbanne Cedex, France. le_zhang@hotmail.com [corrected]
Buchet
René
R
Azzar
Gérard
G
D000469:Alkaline Phosphatase / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D004926:Escherichia coli / Q000201:enzymology*; D008274:Magnesium / Q000378:metabolism*; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D015032:Zinc / Q000378:metabolism*
D000469:Alkaline Phosphatase; D008274:Magnesium; D015032:Zinc