Structural features and dynamics of a cold-adapted alkaline phosphatase studied by EPR spectroscopy.
EPR spectroscopy, performed after site-directed spin-labeling, was used to study structural dynamics in a cold-adapted alkaline phosphatase (EC 3.1.1.1). Differences in the structural environment of six spin-labeled side chains allowed them to be classified (with reference to previously obtained mobility maps) as belonging to loop positions (either relatively surface exposed or in structural contact) or helix positions (surface exposed, in contact, or buried). The mobility map constructed in the present study provides structural information that is in broad agreement with the location in the crystal structure. All but one of the chosen serine-to-cysteine mutations reduced activity considerably and this coincided with improved thermal stability. The effect of spin-labeling on enzyme function ranged from nonperturbing to an almost complete loss of activity. In the latter case, treatment with a thiol reagent reactivated the enzyme, indicating relief of steric hindrance to the catalytic process. Two mutations of an active-site residue W274 (K328 in Escherichia coli alkaline phosphatase), known to reduce activity and increase stability of Vibrio alkaline phosphatase, gave a coincidental reduction in mobility of a nearby spin-label located at C67, as determined by EPR spectroscopy. This suggests that movement of the helix carrying C67 and the closely positioned nucleophilic S65 is interconnected with catalytic events.
Heidarsson
Pétur O
PO
Department of Biochemistry, Science Institute, University of Iceland, Reykjavik, Iceland.
Sigurdsson
Snorri Th
ST
Asgeirsson
Bjarni
B
D000469:Alkaline Phosphatase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003080:Cold Temperature; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013113:Spin Labels; D014733:Vibrio / Q000201:enzymology
D013113:Spin Labels; D000469:Alkaline Phosphatase