Interactions of pyridoxal kinase and aspartate aminotransferase emission anisotropy and compartmentation studies.
Physical interactions between pyridoxal kinase and aspartate aminotransferase were detected by means of emission anisotropy and affinity chromatography techniques. Binding of aspartate aminotransferase (apoenzymes) to pyridoxal kinase tagged with a fluorescent probe was detected by emission anisotropy measurements at pH 6.8 (150 mM KCl). Upon saturation of the kinase with the aminotransferase, the emission anisotropy increases 22%. The protein complex is characterized by a dissociation constant of 3 microM. Time-dependent emission anisotropy measurements conducted with the mixture 5-naphthylamine-1-sulfonic acid-kinase aspartate aminotransferase (apoenzyme), revealed the presence of two rotational correlation times of phi 1 = 36 and phi 2 = 62 ns. The longer correlation time is attributed to the stable protein complex. By immobilizing one enzyme (pyridoxal kinase) through interactions with pyridoxal-Sepharose, it was possible to demonstrate that aspartate aminotransferase releases pyridoxal kinase. A test of compartmentation of pyridoxal-5-phosphate within the protein complex using alkaline phosphatase as trapping agent, indicates that the cofactor generated by the catalytic action of the kinase is channeled to the apotransaminase. The main function of the stable complex formed by the kinase and the aminotransferase is to hinder the release of free pyridoxal-5-phosphate into the bulk solvent.
Kim
Y T
YT
Department of Biochemistry, University of Tennessee, Knoxville 37996-0840.
Kwok
F
F
Churchich
J E
JE
D000818:Animals; D001051:Apoenzymes / Q000378:metabolism; D001219:Aspartate Aminotransferases / Q000378:metabolism*; D001921:Brain / Q000201:enzymology*; D002846:Chromatography, Affinity; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D017879:Isothiocyanates; D007700:Kinetics; D009282:Naphthalenesulfonates; D010770:Phosphotransferases / Q000378:metabolism*; D011731:Pyridoxal Kinase / Q000378:metabolism*; D011732:Pyridoxal Phosphate / Q000378:metabolism; D012756:Sheep; D013053:Spectrophotometry; D013861:Thiocyanates
D001051:Apoenzymes; D005456:Fluorescent Dyes; D017879:Isothiocyanates; D009282:Naphthalenesulfonates; D013861:Thiocyanates; C037152:isothiocyanic acid; D011732:Pyridoxal Phosphate; C006421:1,5-I-AEDANS; D001219:Aspartate Aminotransferases; D010770:Phosphotransferases; D011731:Pyridoxal Kinase; C022042:1-naphthylamine-5-sulfonic acid