title: Reactions of 3'-O-methylpyridoxal 5'-phosphate in aspartate aminotransferase.
abstract: The reaction of 3'-O-methylpyridoxal 5'-phosphate bound into the active site of aspartate aminotransferase with the substrate L-aspartate has been investigated. This methylated coenzyme is a very poor catalyst but it does function slowly to produce normal products of a transamination half-reaction. At pH 8.5 and above the characteristic absorption band of a quinonoid intermediate appears rapidly and becomes very intense when the aspartate concentration is raised to 2 M. At pH 6 the quinonoid band is not seen, but the conversion of the methylated coenzyme into 3'-O-methylpyridoxamine 5'-phosphate is about 7 times faster than at high pH with the pH dependence being determined by an apparent pKa of 8.1 at 30 degrees C. We suggest that the active site containing the methylated coenzyme carries a net charge 1 unit more positive than that of native enzyme. This causes a loss of some other proton from the active site and could leave the catalytic lysine-258 deprotonated in the quinonoid species. This may explain its inability to react rapidly. We have measured the spectral band shapes of the quinonoid species studied here and have compared it with that seen with native enzyme. Because of the close similarity we conclude that during normal transamination the proton bound to the imine nitrogen probably shifts onto the phenolic oxygen prior to or synchronously with the formation of the observed quinonoid species.
authors: Department of Biochemistry and Biophysics, Iowa State University, Ames 50011.
authors: V J
authors: VJ
authors: Chen
authors: D E
authors: DE
authors: Metzler
authors: W T
authors: WT
authors: Jenkins
mesh_terms: D001219:Aspartate Aminotransferases; D001224:Aspartic Acid; D006863:Hydrogen-Ion Concentration; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives*; D013053:Spectrophotometry
chemical_list: C053916:3'-O-methylpyridoxal 5'-phosphate; D001224:Aspartic Acid; D011732:Pyridoxal Phosphate; D001219:Aspartate Aminotransferases