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 th e denaturation and renaturation of the enzyme Escherichia coli alkalin e phosphatase. We show that GTP, ATP, and their nonhydrolyzable analog ues considerably stimulate renaturation of AP in the presence of 2-mer captoethanol where spontaneous renaturation is completely arrested due to reduction of S-S bonds. GTP is the most efficient inducer of recon stitution of the active site and appears to play a specific role besid es being a substrate. The reconstituted protein was found to be in the reduced form despite having near-normal activity. The self-refolded o xidized form and the GTP-refolded reduced form had the same K-M/k(cat) 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 ac tivity of AP. We also report here on the Zn2+-assisted reconstitution of E. coli AP under reducing condition. The prior formation of a disul fide bond for positioning the active site residues in the proper geome try is unnecessary under this condition.