Iodination of glyceraldehyde 3-phosphate dehydrogenase from Bacillus stearothermophilus.
The reaction of iodine with glyceraldehyde 3-phosphate dehydrogenase from Bacillus stearothermophilus was investigated. The active-site thiol group of the cysteine residue homologous with cysteine-149 in the pig muscle enzyme was protected by reaction with tetrathionate. The apoenzyme was readily inhibited by KI3 solution at pH8, but the coenzyme, NAD+, protected the enzyme against inhibition and decreased the extent of iodination. At pH 9.5, ready inhibition of both apo- and holo-enzyme was observed. Tryptic peptides containing residues iodinated at pH 8 were isolated and characterized. One of the most reactive residues in both holo- and apo-enzymes was a tyrosine homologous with tyrosine-46 in the pig muscle enzyme, and this residue was iodinated without loss of enzymic activity. Other reactive tyrosine residues in the apoenzyme were in positions homologous with residues 178, 273, 283 and 311 in the pig muscle enzyme, but they were not readily iodinated in the holoenzyme. Histidine residues in both holo- and apo-enzymes were iodinated at pH 8 in sequence positions homologous with residues 50, 162 and 190 in the pig muscle enzyme. The inhibition of the enzyme was not correlated with the iodination of a particular residue. The results are discussed in relation to a three-dimensional model based on the structure of the lobster muscle enzyme and demonstrate that conformational changes affecting the reactivity of several tyrosine residues most probably occur on binding of the coenzyme.
D000595:Amino Acid Sequence; D001051:Apoenzymes; D004586:Electrophoresis; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000037:antagonists & inhibitors; D006639:Histidine / Q000032:analysis; D007455:Iodine; D007470:Monoiodotyrosine / Q000032:analysis; D009243:NAD; D010446:Peptide Fragments / Q000032:analysis; D011487:Protein Conformation; D013053:Spectrophotometry; D014443:Tyrosine / Q000032:analysis
D001051:Apoenzymes; D010446:Peptide Fragments; D009243:NAD; D014443:Tyrosine; D006639:Histidine; D007455:Iodine; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D007470:Monoiodotyrosine