Engineered disulfide bonds increase active-site local stability and reduce catalytic activity of a cold-adapted alkaline phosphatase.
Alkaline phosphatase is an extracellular enzyme that is membrane-bound in eukaryotes but resides in the periplasmic space of bacteria. It normally carries four cysteine residues that form two disulfide bonds, for instance in the APs of Escherichia coli and vertebrates. An AP variant from a Vibrio sp. has only one cysteine residue. This cysteine is second next to the nucleophilic serine in the active site. We have individually modified seven residues to cysteine that are on two loops predicted to be within a 5 A radius. Four of them formed a disulfide bond to the endogenous cysteine. Thermal stability was monitored by circular dichroism and activity measurements. Global stability was similar to the wild-type enzyme. However, a significant increase in heat-stability was observed for the disulfide-containing variants using activity as a measure, together with a large reduction in catalytic rates (k(cat)) and a general decrease in Km values. The results suggest that a high degree of mobility near the active site and in the helix carrying the endogenous cysteine is essential for full catalytic efficiency in the cold-adapted AP.
Asgeirsson
Bjarni
B
Department of Biochemistry, Science Institute, University of Iceland, Dunhaga 3, IS107 Reykjavík, Iceland. bjarni@raunvis.hi.is
Adalbjörnsson
Björn Vidar
BV
Gylfason
Gudjón Andri
GA
D000469:Alkaline Phosphatase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D003080:Cold Temperature; D004220:Disulfides / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006358:Hot Temperature; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D040681:Structural Homology, Protein; D014733:Vibrio / Q000201:enzymology / Q000235:genetics
D004220:Disulfides; D000469:Alkaline Phosphatase