Limited tryptic proteolysis of pig kidney 3,4-dihydroxyphenylalanine decarboxylase.
Pig kidney 3,4-dihydroxyphenylalanine (Dopa) decarboxylase can be nicked by trypsin with complete loss of its catalytic activity. The original dimer of subunit molecular weight of about 52,000 yields fragments of Mr 38,000 and 14,000, as seen on sodium dodecyl sulfate-gel electrophoresis. Though inactive, the nicked protein retains its native molecular weight and its capacity to bind pyridoxal-5'-phosphate (pyridoxal-P), is recognized by an antiserum raised against the native enzyme, and forms Schiff's base intermediates with aromatic amino acids in L and D forms. Thus, the nicked protein appears to be in a conformation--closely resembling that of the original enzyme--which consists of a tight association of the two tryptic fragments. Dissociation and separation of the two fragments can be achieved under denaturing conditions on a reverse-phase HPLC column. The pyridoxal-P binding site is located on the larger fragment. No NH2-terminal residue is detected in either the intact enzyme or the larger fragment, whereas analysis of the smaller fragment yields a sequence of the first 50 amino acid residues. These data indicate that the smaller fragment is located at about one-third from the COOH terminus of Dopa decarboxylase, while the larger fragment constitutes the aminic portion of the molecule. The site of trypsin cleavage seems to be in a region of the enzyme particularly susceptible to proteolysis. The results of these studies contribute to a better understanding of the structural properties of pig kidney Dopa decarboxylase and may constitute an important step toward the elucidation of the enzyme's primary structure.
D006916:5-Hydroxytryptophan / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D065105:Aromatic Amino Acid Decarboxylase Inhibitors; D001142:Aromatic-L-Amino-Acid Decarboxylases / Q000378:metabolism*; D001665:Binding Sites; D004295:Dihydroxyphenylalanine / Q000494:pharmacology; D004296:Dopa Decarboxylase / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D007163:Immunosorbent Techniques; D007668:Kidney / Q000201:enzymology*; D007980:Levodopa / Q000494:pharmacology; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism; D011487:Protein Conformation; D011732:Pyridoxal Phosphate / Q000378:metabolism; D013053:Spectrophotometry; D013552:Swine; D014357:Trypsin / Q000378:metabolism* / Q000494:pharmacology
D065105:Aromatic Amino Acid Decarboxylase Inhibitors; D046911:Macromolecular Substances; D010446:Peptide Fragments; D007980:Levodopa; D011732:Pyridoxal Phosphate; D004295:Dihydroxyphenylalanine; D006916:5-Hydroxytryptophan; D014357:Trypsin; D004296:Dopa Decarboxylase; D001142:Aromatic-L-Amino-Acid Decarboxylases