REACTION OF APOASPARTATE AMINOTRANSFERASE WITH ANALOGS OF PYRIDOXAL PHOSPHATE

The interactions of a variety of analogs of pyridoxal phosphate with aspartate aminotransferase have been studied in an attempt to investigate the role of the functional groups of pyridoxal phosphate in binding and in catalysis. Pyridoxal phosphate N-oxide, N-methylpyridoxal phosphate, O-methylpyridoxal phosphate, and pyridoxal phosphate analogs which contain modified substituents in the 5 position (CH2CH2COOH, CH2PO3H2, CH2OPO2(CH3)H, and CH(CH3)OPO3H2) were found to bind at the pyridoxal phosphate binding site. This conclusion was based on the observations that (1) the analogs in the presence of apoenzyme have absorption and circular dichroism spectral properties similar to those observed with the holoenzyme. However the pKA of 6.3 for the native enzyme is shifted to below 4 for the analogs bearing carboxyl or methylphosphonate groups in the 5 position; (2) the bound analogs undergo reversible transamination by L-glutamate; and (3) the analogs are displaced slowly from the apoenzyme by pyridoxal phosphate. Only the enzymebound pyridoxal phosphate A-oxide and α-5′-C-methylpyridoxal phosphate show 1% or more of the activity of pyridoxal phosphate. Some possible explanations for the very low activation of the enzyme by most of these analogs, in spite of their apparent normal binding, are discussed. © 1969, American Chemical Society. All rights reserved.