Characterization of the three tyrosine residues of delta 5-3-ketosteroid isomerase by time-resolved fluorescence and circular dichroism.
delta 5-3-Ketosteroid isomerase (EC 5.3.3.1) of Pseudomonas testosteroni converts delta 5-3-ketosteroids to delta 4-3-ketosteroids via an enolic intermediate. Site-specific mutagenesis has identified Tyr-14 and Asp-38 as the catalytically essential general acid and base, respectively. Three tyrosine residues (Tyr-14, Tyr-55, and Tyr-88) are the only significant fluorophores in the wild-type isomerase. Recent studies of the steady-state fluorescence of the wild-type enzyme and all six mutant enzymes in which one or two tyrosine residues have been mutated to phenylalanine show that the fluorescence intensity of Tyr-14 is very high, that of Tyr-88 is very low, and that of Tyr-55 is intermediate and comparable to that of N-acetyltyrosine amide in solution (Li, Y.-K., Kuliopulos, A., Mildvan, A.S., & Talalay, P. (1993) Biochemistry 32, 1816-1824). Extension of these experiments by time-resolved fluorescence and fluorescence anisotropy measurements demonstrates that Tyr-14, which is in a hydrophobic environment, has an unusually long fluorescence lifetime (4.6 ns) as compared to Tyr-55 (2.0 ns) or Tyr-88 (0.8 ns) and to most protein tyrosine residues (0.2-2 ns). The Förster distances obtained from the absorption and emission of these tyrosines predict that total quenching of Tyr-14 fluorescence by Tyr-55, and to a lesser degree by Tyr-88, would occur if their orientations were favorable.(ABSTRACT TRUNCATED AT 250 WORDS)
Wu
P
P
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218.
Li
Y K
YK
Talalay
P
P
Brand
L
L
D002942:Circular Dichroism; D005454:Fluorescence Polarization; D019791:Guanidine; D006146:Guanidines; D006358:Hot Temperature; D016296:Mutagenesis; D011489:Protein Denaturation; D011549:Pseudomonas / Q000201:enzymology; D013251:Steroid Isomerases / Q000737:chemistry*; D014443:Tyrosine / Q000737:chemistry*
D006146:Guanidines; D014443:Tyrosine; D013251:Steroid Isomerases; C022660:steroid delta-isomerase; D019791:Guanidine