Excess nucleoside triphosphates (or zinc) allow recovery of alkaline phosphatase activity following refolding under reducing conditions.
The contribution of ATP and other nucleotides to the stabilization of non-native structures has been described for some proteins. We report here the effect of GTP, ATP, and their nonhydrolyzable analogues on the denaturation and renaturation of the enzyme Escherichia coli alkaline phosphatase. We show that GTP, ATP, and their nonhydrolyzable analogues considerably stimulate renaturation of AP in the presence of 2-mercaptoethanol where spontaneous renaturation is completely arrested due to reduction of S-S bonds. GTP is the most efficient inducer of reconstitution of the active site and appears to play a specific role besides being a substrate. The reconstituted protein was found to be in the reduced form despite having near-normal activity. The self-refolded oxidized form and the GTP-refolded reduced form had the same KM/kcat values and showed similar structural properties. We conclude that GTP can not only induce reconstitution of dimerization-competent monomers because of its substrate nature but also act as a modulator of the activity of AP. We also report here on the Zn2+-assisted reconstitution of E. coli AP under reducing condition. The prior formation of a disulfide bond for positioning the active site residues in the proper geometry is unnecessary under this condition.
Ghosh
N
N
Department of Biochemistry and Biophysics, Kalyani University, India.
Sarkar
S N
SN
Roy
K B
KB
D000255:Adenosine Triphosphate / Q000737:chemistry*; D000469:Alkaline Phosphatase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology; D019791:Guanidine; D006160:Guanosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D017510:Protein Folding; D019163:Reducing Agents; D015032:Zinc / Q000737:chemistry*
D019163:Reducing Agents; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D000469:Alkaline Phosphatase; D015032:Zinc; D019791:Guanidine