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Protein Science (2003), 12:1039-1050.
Copyright © 2003 The Protein Society

Involvement of conserved asparagine and arginine residues from the N-terminal region in the catalytic mechanism of rat liver and Trypanosoma cruzi tyrosine aminotransferases

Verónica R. Sobrado1, Marisa Montemartini-Kalisz2,3, Henryk M. Kalisz2,3, María Candelaria de la Fuente1, Hans-Jürgen Hecht2 and Cristina Nowicki1

1 IQUIFIB (CONICET, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires), Buenos Aires, Argentina
2 GBF, Gesellschaft für Biotechnologische Forschung, Braunschweig, Germany

Reprint requests to: Cristina Nowicki, IQUIFIB (CONICET, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires), Junín 956, CP 1113 Buenos Aires, Argentina; e-mail: cnowicki{at}criba.edu.ar; fax: 54-114-962-5457.

Rat liver and Trypanosoma cruzi tyrosine aminotransferases (TATs) share over 40% sequence identity, but differ in their substrate specificities. To explore the molecular features related to these differences, comparative mutagenesis studies were conducted on full length T. cruzi TAT and N-terminally truncated rat TAT recombinant enzymes. The functionality of Arg315 and Arg417 in rat TAT was investigated for comparison with the conserved Arg292 and Arg386 in aspartate and bacterial aromatic aminotransferases (ASATs and ARATs). The rat TAT Arg315Lys variant remained fully active indicating that, as in T. cruzi TAT and contrary to subfamily I{alpha} aminotransferases, this residue is not critical for activity. In contrast, the Arg417Gln variant was inactive. The catalytic relevance of the putative rat TAT active site residues Asn54 and Arg57, which are strictly conserved in TATs (Asn17 and Arg20 in T. cruzi TAT) but differ in ASATs and ARATs, was also explored. The substitutions Arg57Ala and Arg57Gln abolished enzymatic activity of these mutants. In both variants, spectral studies demonstrated that aromatic but not dicarboxylic substrates could efficiently bind in the active site. Thus, Arg57 appears to be functionally equivalent to Arg292 of ASATs and ARATs. Asn54 also appears to be involved in the catalytic mechanism of rat TAT since its exchange for Ser lowered the kcat/Km ratios towards its substrates. Mutation of the analogous residues in T. cruzi TAT also lowered the catalytic efficiencies (kcat/Km) of the variants substantially. The results imply that the mamalian TAT is more closely related to the T. cruzi TAT than to ASATs and ARATs.

Keywords: Aromatic aminotransferases; rat liver tyrosine aminotransferase; heterologous expression; site directed mutagenesis; Trypanosoma cruzi


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