Effects of Replacing Active Site Residues in a Cold-Active Alkaline Phosphatase With Those Found in Its Mesophilic Counterpart From Escherichia Coli

FEBS J. 2008 Jan;275(1):117-27. doi: 10.1111/j.1742-4658.2007.06182.x. Epub 2007 Dec 7.

Abstract

Alkaline phosphatase (AP) from a North Atlantic marine Vibrio bacterium was previously characterized as being kinetically cold-adapted. It is still unknown whether its characteristics originate locally in the active site or are linked to more general structural factors. There are three metal-binding sites in the active site of APs, and all three metal ions participate in catalysis. The amino acid residues that bind the two zinc ions most commonly present are conserved in all known APs. In contrast, two of the residues that bind the third metal ion (numbered 153 and 328 in Escherichia coli AP) are different in various APs. This may explain their different catalytic efficiencies, as the Mg2+ most often present there is important for both structural stability and the reaction mechanism. We have mutated these key residues to the corresponding residues in E. coli AP to obtain the double mutant Asp116/Lys274, and both single mutants. All these mutants displayed reduced substrate affinity and lower overall reaction rates. The Lys274 and Asp116/Lys274 mutants also displayed an increase in global heat stability, which may be due to the formation of a stabilizing salt bridge. Overall, the results show that a single amino acid substitution in the active site is sufficient to alter the structural stability of the cold-active Vibrio AP both locally and globally, and this influences kinetic properties.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptation, Biological
  • Alkaline Phosphatase / chemistry*
  • Alkaline Phosphatase / genetics
  • Alkaline Phosphatase / metabolism
  • Binding Sites
  • Catalysis
  • Escherichia coli / enzymology
  • Kinetics
  • Models, Molecular
  • Protein Denaturation
  • Protein Folding
  • Protein Structure, Secondary
  • Temperature
  • Urea / metabolism
  • Urea / pharmacology
  • Vibrio / classification
  • Vibrio / enzymology*
  • Vibrio / metabolism
  • Zinc

Substances

  • Urea
  • Alkaline Phosphatase
  • Zinc