1Y6V: Structure Of E. Coli Alkaline Phosphatase In Presence Of Cobalt At 1.60 A Resolution

Escherichia coli alkaline phosphatase exhibits maximal activity when Zn(2+) fills the M1 and M2 metal sites and Mg(2+) fills the M3 metal site. When other metals replace the zinc and magnesium, the catalytic efficiency is reduced by more than 5000-fold. Alkaline phosphatases from organisms such as Thermotoga maritima and Bacillus subtilis require cobalt for maximal activity and function poorly with zinc and magnesium. Previous studies have shown that the D153H alkaline phosphatase exhibited very little activity in the presence of cobalt, while the K328W and especially the D153H/K328W mutant enzymes can use cobalt for catalysis. To understand the structural basis for the altered metal specificity and the ability of the D153H/K328W enzyme to utilize cobalt for catalysis, we determined the structures of the inactive wild-type E. coli enzyme with cobalt (WT_Co) and the structure of the active D153H/K328W enzyme with cobalt (HW_Co). The structural data reveal differences in the metal coordination and in the strength of the interaction with the product phosphate (P(i)). Since release of P(i) is the slow step in the mechanism at alkaline pH, the enhanced binding of P(i) in the WT_Co structure explains the observed decrease in activity, while the weakened binding of P(i) in the HW_Co structure explains the observed increase in activity. These alterations in P(i) affinity are directly related to alterations in the coordination of the metals in the active site of the enzyme.
PDB ID: 1Y6VDownload
MMDB ID: 33706
PDB Deposition Date: 2004/12/7
Updated in MMDB: 2012/10
Experimental Method:
x-ray diffraction
Resolution: 1.6  Å
Source Organism:
Similar Structures:
Biological Unit for 1Y6V: dimeric; determined by author and by software (PISA)
Molecular Components in 1Y6V
Label Count Molecule
Proteins (2 molecules)
Alkaline Phosphatase(Gene symbol: phoA)
Molecule annotation
Chemicals (10 molecules)
* Click molecule labels to explore molecular sequence information.

Citing MMDB