Biochemical characterization of aspartate aminotransferase allozymes from common wheat

Six allozymes of aspartate aminotransferase (AAT, EC 2.6.1.1): three plastidial (AAT-2 zone) and three cytosolic (AAT-3 zone) were isolated from common wheat (Triticum aestivum) seedlings and highly purified by a five-step purification procedure. The identity of the studied proteins was confirmed by mass spectrometry. The molecular weight of AAT allozymes determined by gel filtration was 72.4±3.6 kDa. The molecular weights of plastidial and cytosolic allozymes estimated by SDS-PAGE were 45.3 and 43.7 kDa, respectively. The apparent Michaelis constant (K _m) values determined for four substrates appeared to be very similar for each allozyme. The values of the turnover number (k _cat) and the k _cat/K _m ratio calculated for allozymes with L-aspartate as a leading substrate were in the range of 88.5–103.8 s^?1/10,412–10,795 s^?1 M^?1 for AAT-2 zone and 4.6–7.0 s^?1/527–700 s^?1 M^?1 for AAT-3 zone. These results clearly demonstrated much higher catalytic efficiency of AAT-2 allozymes. Therefore, partial sequences of cDNA encoding AATs from different zones were obtained using the RT-PCR technique. Comparison of the AAT-2 and AAT-3 amino acid sequences from active site regions revealed five non-conservative substitutions, which impact on the observed differences in the isozymes catalytic efficiency is discussed.