Biochemical characterization of aspartate aminotransferase allozymes from common wheat

Abstract

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. © 2013 Versita Warsaw and Springer-Verlag Wien.