Mutation of aspartate-233 to valine in mouse ornithine decarboxylase reduces enzyme activity.
Ornithine decarboxylase is the first and key enzyme in mammalian polyamine biosynthesis. All eukaryotic ornithine decarboxylases contain several highly conserved regions and the amino acid residues 232-238 form one of the most highly conserved sequences. This region contains a glycine-rich sequence typically found in a number of pyridoxal 5'-phosphate-dependent or nucleotide-binding proteins. We mutated aspartate-233 which is the only acidic residue within this region to valine. This mutation causes striking sequence similarity with the guanine nucleotide binding domain of c-H-ras. Mutated ornithine decarboxylase cDNA with a mouse mammary tumor virus long terminal repeat promoter has been transfected for stable expression into ornithine decarboxylase-deficient C55.7 cells. Ornithine decarboxylase activity of the mutated enzyme was about 20% of wild-type ornithine decarboxylase activity and it was not activated by guanosine triphosphate like the ornithine decarboxylase isoform found in some tumors and rat brain. The mutation caused an increase in K(m) value of about 20-fold both for the substrate L-ornithine and for the cofactor pyridoxal 5'-phosphate. The Ki value for the irreversible inhibitor alpha-difluoromethylornithine was also increased, whereas the half-life of the enzyme was shortened. These results suggest that the region containing aspartate-233 is essential for binding of the cofactor and thus forms part of enzymatic active site, and the mutation of aspartate-233 to valine cannot, at least alone, cause the activation of ornithine decarboxylase by guanosine triphosphate (230).
Kilpeläinen
P T
PT
Department of Biochemistry, University of Oulu, Finland.
Hietala
O A
OA
D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D016466:CHO Cells; D017124:Conserved Sequence; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects; D006160:Guanosine Triphosphate / Q000494:pharmacology; D007700:Kinetics; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009955:Ornithine Decarboxylase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D051381:Rats; D014162:Transfection; D014633:Valine / Q000737:chemistry / Q000235:genetics
D018076:DNA, Complementary; D001224:Aspartic Acid; D006160:Guanosine Triphosphate; D009955:Ornithine Decarboxylase; D014633:Valine