Structure/function relationship studies on the T/S residues 173-177 of rat ODC.
A well-conserved T/S cluster was detected among vertebrate ornithine decarboxylase by computer analysis (E. Viguera, O. Trelles, J.L. Urdiales, J.M. Matés, F. Sánchez-Jiménez, Trends Biochem. Sci. 19 (1994) 318-319). In the present report we studied the role of these residues (173, 176 and 177 in rat ornithine decarboxylase (ODC)) in enzymic activity 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 the negative charged fragment of T. brucei ODC (KVEDC) did not affect protein stability, but increased Km values of the mutant enzyme. The substitution of the T/S residues by alanine also has a similar effect on rat ODC kinetic values. However, results indicate that polarity of the fragment must be an important factor for protein conformation, since the latter mutant, having no T/S or D/E residue in the fragment (ALKAA), showed reduced stability in vitro.
Matés
J M
JM
Laboratorio de Bioquímica y Biología Molecular, Facultad de Ciencias/Instituto de Biotecnología, Universidad de Málaga, Campus de Teatinos, 29071 Malaga, Spain.
del Valle
A E
AE
Urdiales
J L
JL
Coleman
C S
CS
Feith
D
D
Olmo
M T
MT
Pegg
A E
AE
Sánchez-Jiménez
F
F
D000595:Amino Acid Sequence; D000818:Animals; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D004795:Enzyme Stability; D005057:Eukaryotic Cells; D008322:Mammals; D009154:Mutation; D009955:Ornithine Decarboxylase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine; D013329:Structure-Activity Relationship; D013912:Threonine; D014346:Trypanosoma brucei brucei / Q000201:enzymology
D010446:Peptide Fragments; D011994:Recombinant Proteins; D013912:Threonine; D012694:Serine; D009955:Ornithine Decarboxylase