Rotational dynamics of 4-aminobutyrate aminotransferase.
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 Kd of 6 microM. 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 (phi = 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 31P-NMR at 72.86 MHz. In the native enzyme, the pyridoxal-5-P 31P-chemical shift is pH-independent, indicating that the phosphate group is well protected from the solvent. The correlation time determined from the 31P-spectrum of the aminotransferase exceeds the value calculated for the hydrated spherical model (phi = 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.
D000612:4-Aminobutyrate Transaminase / Q000378:metabolism*; D000817:Anilino Naphthalenesulfonates / Q000494:pharmacology; D000818:Animals; D001921:Brain / Q000201:enzymology; D005456:Fluorescent Dyes / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013552:Swine; D000637:Transaminases / Q000378:metabolism*
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; C027132:1-anilino-8-naphthalenesulfonate; D000637:Transaminases; D000612:4-Aminobutyrate Transaminase