Mammalian S-adenosylmethionine decarboxylase was expressed at a high level in an Escherichia coli mutant deficient in this enzyme. The proenzyme form of this enzyme was cleaved and processed to the mature decarboxylase which contains two pairs of nonidentical subunits, the larger of which contains a pyruvate prosthetic group. In order to determine the site of formation of the pyruvate, two approaches were used. First, the mammalian S-adenosylmethionine decarboxylase produced in E. coli was purified to homogeneity and the pyruvate converted to alanine by a reductive amination. The large subunit was then isolated by reversed phase high pressure liquid chromatography and the amino-terminal sequence determined and compared with the sequence of the proenzyme derived from its cDNA. These results indicated that the bond between glutamic acid 67 and serine 68 was the site of cleavage. Second, each of the serine residues in portion of the proenzyme likely to contain the cleavage site were altered by site-directed mutagenesis and the RNA produced from plasmids containing these mutations was translated in a reticulocyte lysate. The translation products were tested for processing and for S-adenosylmethionine decarboxylase activity. Altering the serine residues at positions 50, 66, and 69 to alanines had little effect but changing serine at position 68 to alanine completely prevented both processing and activity. These results indicate that the serine residue at position 68 of the proenzyme which is in the underlined position in the sequence -Leu-Ser-Glu-Ser-Ser-Met- is the residue which is converted to the pyruvate prosthetic group in human S-adenylmethionine decarboxylase.
|Number of pages
|Journal of Biological Chemistry
|Published - 1989
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology