Fluorescence and chemical modification of tryptophan as probes of structure of GTP:AMP phosphotransferase from beef heart mitochondria.
Selective modification of the two Trp residues of GTP:AMP phosphotransferase from beef heart mitochondria (Mr 26 000; MgGTP + AMP in equilibrium MgGDP + ADP) has been attained by treatment of the enzyme with N-bromosuccinimide at pH 4.0. Almost complete loss of activity is observed when one Trp is oxidized. Fluorescence emission spectra (lambda exc 295 nm) were recorded over the pH range 1.9-12.2. Quenching constants, K, with acrylamide were 4.9, 3.4, 3.1, 2.4, 9.2 and 9.4 M-1 at respective pH values of 11.1, 7.5, 5.5, 4.0, 1.9 and 7.5 with 6 M guanidine/HCl. Over the pH range 8.0-5.5 the fluorescence peak has a constant height with maximum at 333-334 nm, which can be segregated by acrylamide quenching into a peak with maximum at 338 nm and another with maximum at 330 nm. Dropping the pH from 5.5 to 4.0 results in the fluorescence at 338 nm decreasing to 335 nm (indicative of less exposure of the Trp) while that at 330 nm remains constant. Thus the limitation of reactivity to N-bromosuccinimide to pH 4.0 or lower cannot be accounted for by increased exposure of the Trp residues but rather must be explained by a change in the microenvironment of each Trp. As shown by K values above, at pH 2.0 Trp residues are exposed to the solvent, as in the case of treatment with 6 M guanidine hydrochloride. In raising the pH from 8.0 to 12.0 a number of changes occur: (a) the lambda max of emission shifts from 333-334 nm to 343 nm; (b) residue(s) become(s) more available to acrylamide quenching; (c) fluorescence decreases and enzymatic activity increases, both with a midpoint at about 10.6; (d) absorption difference spectra show a maximum at 295 nm typical of Tyr ionization. These data are consistent with conformational change as the pH becomes more alkaline making the Trp residue(s) more exposed to the solvent and/or to non-radiative energy transfer to tyrosinate.
D000818:Animals; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D006863:Hydrogen-Ion Concentration; D008929:Mitochondria, Heart / Q000201:enzymology*; D009703:Nucleoside-Phosphate Kinase; D010084:Oxidation-Reduction; D010770:Phosphotransferases; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000032:analysis*
D014364:Tryptophan; D010770:Phosphotransferases; C021894:nucleoside triphosphate-adenylate kinase; D009703:Nucleoside-Phosphate Kinase