J Mol Evol (1986) 23:328-335 Journal of Molecular Evolution (~ Springer-Verlag NewYorkInc. 1986 The Complete Amino Acid Sequences of Cytosolic and Mitochondrial Aspartate Aminotransferases from Horse Heart, and Inferences on Evolution of the Isoenzymes Shawn Doonan, 1 Filippo Martini, 2 Sebastiana Angelaccio, 2 Stefano Pascarella, 2 Donatella Barra, 2 and Francesco Bossa 2 Department of Biochemistry, University College, Cork, Ireland 2 Dipartimento di Scienze Biochimiche e Centro di Biologia Molecolare del CNR, Universit~ di Roma, Citt~t Universitaria, 00185 Rome, Italy Summary. We report here the complete amino acid sequences of the cytosolic and mitochondrial as- partate aminotransferases from horse heart. The two sequences can be aligned so that 48.1% of the amino acid residues are identical. The sequences have been compared with those of the cytosolic isoenzymes from pig and chicken, the mitochondrial isoen- zymes from pig, chicken, rat, and human, and the enzyme from Escherichia coil The results suggest that the mammalian cytosolic and mitochondrial isoenzymes have evolved at equal and constant rates whereas the isoenzymes from chicken may have evolved somewhat more slowly. Based on the rate of evolution of the mammalian isoenzymes, the gene- duplication event that gave rise to cytosolic and mitochondrial aspartate aminotransferases is esti- mated to have occurred at least 109 years ago. The cytosolic and mitochondrial isoenzymes are equally related to the enzyme from E. coli; the prokaryotic and eukaryotic enzymes diverged from one another at least 1.3 • 109 years ago. Key words: Aspartate aminotransferase -- Isoen- zyme -- Protein evolution -- Mitochondria Offprint requests to: S. Doonan Introduction The cytosolic and mitochondrial isoenzymes of as- partate aminotransferase (EC 2.6.1.1) are coded for by different, but structurally related, genes. This was first shown conclusively by comparisons of the ami- no acid sequences of the two isoenzymes from pig heart (Barra et al. 1980)--indeed this was the first pair of cytosolic and mitochondrial isoenzymes for which complete amino acid sequences were estab- lished. Aspartate aminotransferases have been the ob- jects of extensive structural studies in recent years. This interest stems at least in part from the fact that the mitochondrial isoenzyme is synthesized in the ceil cytosol and then translocated through the mi- tochondrial membrane system into the matrix; translocation is a posttranslational event (for a re- view of these processes, see Doonan et al. 1984a). It seems logical to postulate that the requirement for translocation through the mitochondrial mem- brane system has imposed extra constraints on the structure of the mitochondrial isoenzyme and that these would be manifest in a slower rate of evolution for this form than for the cytosolic isoenzyme. Com- parative studies of aspartate aminotransferases us- ing immunochemical methods suggested that this was indeed the case (Sonderegger et al. 1977; Porter et al. 1981a), and it was claimed that the mum-