Enzymatic and fluorescence studies of four single‐tryptophan mutants of rat testis fructose 6‐phosphate,2‐kinase:fructose 2,6‐bisphosphatase

Abstract

In order to determine environments around four tryptophan residues, located in the N-terminus, in the kinase and in the phosphatase domains of rat testis Fru 6-P,Z-kinase:Fru 2,6-bisphosphatase, mutant enzymes containing a single tryptophan were constructed by site-directed mutagenesis. 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 . 5~ that of the wild-type enzyme, and Trp 299, Trp 64, Trp 15, and Trp 320 contributed 38%, 28%, 17%, and 17%, respectively. The fluorescence polarization of the wild-type enzyme was significantly lower than any of the mutant enzymes, suggesting proximity of two tryptophan residues in the wild-type enzyme. The polarization in the presence of Fru 6-P affected only Trp 15, which suggested that it is located near the Fru 6-P binding site, but Trp 64 is not. Inactivation of both enzyme activities and unfolding of these enzymes in guanidine 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. Enzymes 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 accessible and that other Trp residues were only slightly accessible to solvent. These results suggest that all the Trp residues are in heterogeneous environments and that none are exposed on the protein surface.

Reaction Mechanism of Fructose-2,6-bisphosphatase

“…The W 3 F mutations did not alter kinetic properties of either Fru-6-P,2-kinase or Fru-2,6-Pase activities significantly (11).…”

“…The cDNA encoding rat testis Fru-6-P,2-kinase/Fru-2,6-Pase (RT2K) was prepared as reported previously (10). The cDNA encoding the Trp-less mutant (RT2K-Wo), in which all four Trp residues of RT2K have been altered to Phe by site-directed mutagenesis was prepared as described previously (11). The pT7-7 RNA polymerase/ promoter plasmid (12) was a gift from S. Tabor (Harvard Medical School).…”

Crystal Structure of the H256A Mutant of Rat Testis Fructose-6-phosphate,2-kinase/Fructose-2,6-bisphosphatase

“…Protein Preparation and Crystallization-The preparation and purification of the Wo form of RT2K was described previously (22,23). A single point mutation of the Wo enzyme, converting His 256 to Ala was generated and purified by the same procedure (1).…”

Introduction of a unique tryptophan residue into various positions of Bacillus licheniformis DnaK

“…These demands are often difficult to satisfy because of the bulky and hydrophobic nature of most fluorescent labels [52,53]. For this reason, numerous water soluble proteins have been studied through their tryptophans [54][55][56][57][58], which are excellent fluorescent probes of structure and dynamics. Interpretation of the fluorescence data is especially straightforward if only one tryptophan residue is present, a feature that can nowadays be achieved routinely using site-directed mutagenesis.…”