TYROSINE QUENCHING OF TRYPTOPHAN PHOSPHORESCENCE IN GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE FROM BACILLUS-STEAROTHERMOPHILUS

Tyrosine is known to quench the phosphorescence of free tryptophan der ivatives in solution, but the interaction between tryptophan residues in proteins and neighboring tyrosine side chains has not yet been demo nstrated. This report examines the potential role of Y283 in quenching the phosphorescence emission of W310 of glyceraldehyde-3-phosphate de hydrogenase from Bacillus stearothermophilus by comparing the phosphor escence characteristics of the wild-type enzyme to that of appositely designed mutants in which either the second tryptophan residue, W84, i s replaced with phenylalanine or Y283 is replaced by valine. Phosphore scence spectra and lifetimes in polyol/buffer low-temperature glasses demonstrate that W310, in both wild-type and W84F (Trp(84) --> Phe) mu tant proteins, is already quenched in viscous low-temperature solution s, before the onset of major structural fluctuations in the macromolec ule, an anomalous quenching that is abolished with the mutation Y283V 9Tyr(283) --> Val). In buffer at ambient temperature, the effect of re placing Y283 with valine on the phosphorescence of W310 is to lengthen its lifetime from 50 mu s to 2.5 ms, a 50-fold enhancement that again emphasizes how W310 emission is dominated by the local interaction wi th Y283. Tyr quenching of W310 exhibits a strong temperature dependenc e, with a rate constant k(q) = 0.1 s(-1) at 140 K and 2 x 10(4) s(-1) at 293 K. Comparison between thermal quenching profiles of the W84F mu tant in solution and in the dry state, where protein flexibility is dr astically reduced, shows that the activation energy of the quenching r eaction is rather small, E-a less than or equal to 0.17 kcal mol(-1), and that, on the contrary, structural fluctuations play an important r ole on the effectiveness of Tyr quenching. Various putative quenching mechanisms are examined, and the conclusion, based on the present resu lts as well as on the phosphorescence characteristics of other protein systems, is that Tyr quenching occurs through the formation of an exc ited-state triplet exciplex.