Tryptophan phosphorescence as a monitor of the solution structure of phosphoglycerate kinase from yeast.
The enzyme phosphoglycerate kinase from yeast possesses two tryptophan residues whose phosphorescence spectrum in low-temperature glasses is resolved into two distinct components with 0-0 vibronic bands centered at 408 and 412.5 nm. The thermal profile of the phosphorescence intensity and lifetime shows that the red (longer wavelength) component is quenched in fluid solutions so that the long-lived phosphorescence observed at ambient temperature in buffer is due entirely to the blue (shorter wavelength) component. The remarkable heterogeneity in flexibility of the two chromophores' sites inferred from the thermal behaviour, when analyzed in terms of the crystallographic structure, allows to make a straightforward assignment of the long-lived emission to internal Trp-333. Because in buffer the phosphorescence is due to only one Trp residue the biphasic nature of the decay reveals the presence of stable, slowly interconverting, conformers with profound differences in the internal fluidity of the C-domain. Further, according to the triplet lifetime, complex formation with substrates affect the protein structure in a very selective way. Thus, while 3-phosphoglycerate has practically no influence on the average lifetime, Mg ATP and Mg ADP increases tau by a factor of 1.9 and 5.3, respectively. The change in lifetime implies a remarkable stiffening of the C-domain which is partly relaxed in ternary complexes with 3-phosphoglycerate. These findings are discussed in terms of ligand-induced "closed" conformations of the protein.
Cioni
P
P
C.N.R.-Istituto di Biofisica, Pisa, Italy.
Puntoni
A
A
Strambini
G B
GB
D002021:Buffers; D008163:Luminescent Measurements; D010735:Phosphoglycerate Kinase / Q000737:chemistry*; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012996:Solutions; D013696:Temperature; D014364:Tryptophan / Q000737:chemistry*
D002021:Buffers; D012996:Solutions; D014364:Tryptophan; D010735:Phosphoglycerate Kinase