STRUCTURE FUNCTION RELATIONSHIP STUDIES ON THE T/S RESIDUE-173-177 OFRAT ODC/

A well-conserved T/S cluster was detected among vertebrate ornithine d ecarboxylase by computer analysis (E. Viguera, O. Trelles, J.L. Urdial es, J.M. Mates, F. Sanchez-Jimenez, Trends Biochem. Sci. 19 (1994) 318 -319). In the present report we studied the role of these residues (17 3, 176 and 177 in rat ornithine decarboxylase (ODC)) in enzymic activi ty and stability by in vitro expression, kinetic characterization and in vitro degradation of site-directed mutants. These T/S residues are substituted by a D/E-enriched fragment in other lower eukaryotic ODCs. The substitution of the T/S-enriched fragment (TLKTS) of rat ODC by t he negative charged fragment of T. brucei ODC (KVEDC) did not affect p rotein stability, but increased K-m values of the mutant enzyme. The s ubstitution of the T/S residues by alanine also has a similar effect o n rat ODC kinetic values. However, results indicate that polarity of t he fragment must be an important factor for protein conformation, sinc e the latter mutant, having no T/S or D/E residue in the fragment (ALK AA), showed reduced stability in vitro. (C) 1998 Elsevier Science B.V. All rights reserved.