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Rotational dynamics of 4‐aminobutyrate aminotransferase
Author(s) -
Churchich Jorge E.,
Kim Doo Sik,
Schnackerz Klaus D.
Publication year - 1983
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(83)80971-5
Subject(s) - chemistry , pyridoxal phosphate , pyridoxal , rotational correlation time , enzyme , fluorescence , molecule , nanosecond , crystallography , active site , fluorescence anisotropy , stereochemistry , biochemistry , cofactor , organic chemistry , quantum mechanics , membrane , optics , laser , physics
The fluorescence dye 1‐anilinonaphthalene‐8‐sulfonate (ANS) was used as a probe of non‐polar binding sites in 4‐aminobutyrate aminotransferase. ANS binds to a single binding site of the dimeric protein with a K d of 6 μM. Nanosecond emission anisotropy measurements were performed on the ANS‐enzyme in an effort to detect independent rotation of the subunits in the native enzyme. The observed rotational correlation time (φ = 65 ns) corresponds to the rotation of a rather rigid dimeric structure. The microenvironment surrounding the natural probe pyridoxal‐5‐P covalently bound to the dimeric structure was explored using 31 P‐NMR at 72.86 MHz. In the native enzyme, the pyridoxal‐5‐P 31 P‐chemical shift is pH‐independent, indicating that the phosphate group is well protected from the solvent. The correlation time determined from the 31 P‐spectrum of the aminotransferase exceeds the value calculated for the hydrated spherical model (φ = 40 ns). It is concluded that the phosphate of the pyridoxal‐5‐P molecule is rigidly bound to the active site of 4‐aminobutyrate aminotransferase.