Effects of ligands on the mobility of an active-site loop in tyrosine hydroxylase as monitored by fluorescence anisotropy.
Fluorescence anisotropy has been used to monitor the effect of ligands on a mobile loop over the active site of tyrosine hydroxylase. Phe184 in the center of the loop was mutated to tryptophan, and the three native tryptophan residues were mutated to phenylalanine to form an enzyme with a single tryptophan residue in the mobile loop. The addition of 6-methyl-5-deazatetrahydropterin to the enzyme resulted in a significant increase in the fluorescence anisotropy. The addition of phenylalanine did not result in a significant change in the anisotropy in the presence or absence of the deazapterin. The K(d) value for the deazapterin was unaffected by the presence of phenylalanine. Qualitatively similar results were obtained with apoenzyme, except that the addition of phenylalanine led to a slight decrease in anisotropy. Frequency-domain lifetime measurements showed that the distribution of lifetimes was unaffected by both the amino acid and deazapterin. Frequency-domain anisotropy analyses were consistent with a decrease in the motion of the sole tryptophan in the presence of the deazapterin. This could be modeled as a decrease in the cone angle for the indole ring of about 12 degrees . The data are consistent with a model in which binding of a tetrahydropterin results in a change in the conformation of the surface loop required for proper formation of the amino acid binding site.
Sura
Giri R
GR
Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843-2128, USA.
Lasagna
Mauricio
M
Gawandi
Vijay
V
Reinhart
Gregory D
GD
Fitzpatrick
Paul F
PF
D019943:Amino Acid Substitution; D016880:Anisotropy; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular; D005453:Fluorescence; D008856:Microscopy, Fluorescence; D009154:Mutation; D010649:Phenylalanine / Q000737:chemistry / Q000235:genetics; D011487:Protein Conformation; D011622:Pterins / Q000737:chemistry; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics; D014446:Tyrosine 3-Monooxygenase / Q000737:chemistry* / Q000235:genetics
D011622:Pterins; C076048:6-methyl-5-deazatetrahydropterin; D010649:Phenylalanine; D014364:Tryptophan; D014446:Tyrosine 3-Monooxygenase