ENZYMATIC AND FLUORESCENCE STUDIES OF 4 SINGLE-TRYPTOPHAN MUTANTS OF RAT TESTIS FRUCTOSE-6-PHOSPHATE,2-KINASE FRUCTOSE-2,6-BISPHOSPHATASE/

In order to determine environments around four tryptophan residues, lo cated in the N-terminus, in the kinase and in the phosphatase domains of rat testis Fru 6-P,2-kinase:Fru 2,6-bisphosphatase, mutant enzymes containing a single tryptophan were constructed by site-directed mutag enesis. The kinetic constants of these mutant enzymes were similar to those of the wild-type enzyme. The sum of the fluorescence intensities of the enzymes was 1.5x that of the wild-type enzyme, and Trp 299, Tr p 64, Trp 15, and Trp 320 contributed 38%, 28%, 17%, and 17%, respecti vely. The fluorescence polarization of the wild-type enzyme was signif icantly lower than any of the mutant enzymes, suggesting proximity of two tryptophan residues in the wild-type enzyme. The polarization in t he presence of Fru 6-P affected only Trp 15, which suggested that it i s located near the Fru 6-P binding site, but Trp 64 is not. Inactivati on of both enzyme activities and unfolding of these enzymes in guanidi ne were monitored by activity assays and fluorescence intensities and maxima. Both Fru 6-P,2-kinase and Fru 2,6-bisphosphatase activities of all these enzymes were inactivated between 0.7 and 1 M guanidine. Enz ymes containing Trp 64 or Trp 15 showed biphasic fractional unfolding curves, but those of Trp 299 or Trp 320 showed gradual steady changes. Fluorescence quenching by iodide indicated that Trp 64 was not access ible and that other Trp residues were only slightly accessible to solv ent. These results suggest that all the Trp residues are in heterogene ous environments and that none are exposed on the protein surface.