Primary structure of human triosephosphate isomerase.
Human placental triosephosphate isomerase was isolated by an improved procedure and recovered with the highest specific activity ever reported. Employing this purification procedure, sufficient amounts of the enzyme were obtained for detailed primary structural studies. For sequences analysis, the enzyme was reduced and carboxymethylated and subjected to tryptic and chymotryptic digestions. The peptide mixtures were separated by high-performance liquid chromatography using octyl or alkylphenyl reverse-phase columns and trifluoroacetic acid/acetonitrile gradient elution systems. Sequence analyses of the intact enzyme, tryptic, chymotryptic, and cyanogen bromide peptides were accomplished using high-sensitivity solid-phase sequencing procedures with either 4-N,N-dimethylaminoazobenzene-4'-isothiocyanate or phenylisothiocyanate. The primary structure of human triosephosphate isomerase is constructed from the alignment of the tryptic peptides with the analysis of the overlapping chymotryptic peptides. The enzyme is a dimeric molecule consisting of two identical polypeptide chains with 248 amino acid residues and a calculated subunit molecular mass of 26,750 daltons. A comparison of the amino acid sequences from the human placental enzyme and from other species such as rabbit, chicken, and coelacanth muscles showed relatively high sequence homology, indicating that the evolution of the enzyme is very conservative. The amino acids of the active-site pocket and the subunit-subunit contact sites exhibit few changes.
D000595:Amino Acid Sequence; D001120:Arginine / Q000032:analysis; D002238:Carbohydrate Epimerases / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D005260:Female; D006801:Humans; D008239:Lysine / Q000032:analysis; D010446:Peptide Fragments / Q000032:analysis; D010920:Placenta / Q000201:enzymology; D011247:Pregnancy; D014305:Triose-Phosphate Isomerase / Q000032:analysis* / Q000302:isolation & purification; D014357:Trypsin / Q000378:metabolism
D010446:Peptide Fragments; D001120:Arginine; D014357:Trypsin; D002238:Carbohydrate Epimerases; D014305:Triose-Phosphate Isomerase; D008239:Lysine