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␣ 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 k cat /K m ratios towards its substrates. Mutation of the analogous residues in T. cruzi TAT also lowered the catalytic efficiencies (k cat /K m ) 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 Aminotransferases belong to the ␣-family of vitamin B 6 -dependent enzymes (Alexander et al. 1994), which play important roles in amino acid biosynthesis and degradation and carbohydrate metabolism in both prokaryotes and eukaryotes. These enzymes possess varied substrate specificities that mainly involve the three 2-oxoacids generated during glycolytic and respiratory cycles. Based on extensive structural, kinetic and site-directed mutagenesis studies on aspartate aminotransferases (ASATs), a ping-pong Bi-Bi mechanism involving several intermediates was proposed for the reversible transamination reaction. Furthermore, the substrate recognition mechanism of ASATs is the best understood among aminotransferases (Kirsch et al. 1984;Arnone et al. 1985;Hayashi 1995).At the structural level, two different aminotransferases belonging to subfamily I␣, the highly specific mammalian

We have previously shown that plasma membrane calcium ATPase (PMCA) pump activity is affected by the membrane protein concentration (Vanagas et al., Biochim Biophys Acta 1768:1641–1644, 2007). Results show evidences for the involvement of the actin cytoskeleton. In this study, we explored the relationship between the polymerization state of actin and its effects on purified PMCA activity. Our results show that PMCA associates with the actin cytoskeleton and this interaction causes a modulation of the catalytic activity involving the phosphorylated intermediate of the pump. The state of actin polymerization determines whether it acts as an activator or an inhibitor of the pump: G-actin and/or short oligomers activate the pump, while F-actin inhibits it. The effects of actin on PMCA are the consequence of direct interaction as demonstrated by immunoblotting and cosedimentation experiments. Taken together, these findings suggest that interactions with actin play a dynamic role in the regulation of PMCA-mediated Ca2+ extrusion through the membrane. Our results provide further evidence of the activation–inhibition phenomenon as a property of many cytoskeleton-associated membrane proteins where the cytoskeleton is no longer restricted to a mechanical function but is dynamically involved in modulating the activity of integral proteins with which it interacts.