Photodependent incorporation of arylazido-beta-alanyl-NAD+ into the coenzyme binding site of yeast glyceraldehyde-3-phosphate dehydrogenase.
Yeast glyceraldehyde-3-phosphate dehydrogenase was labeled in a photodependent reaction by the arylazido-beta-alanyl derivative of NAD+. This analogue was bound covalently to the enzyme and could be reduced in situ by the substrate glyceraldehyde-3-phosphate. That this derivative was bound to the active site in the proper orientation was shown by fluorescence experiments, from the retention of the enzymatic activity when the photolysis of the enzyme-analogue binary complex was carried out in the presence of NAD+. In the dark a non-photodependent competitive inhibition corresponding to a KI-value of 150 microM was observed. Thiol groups of the enzyme were not modified in the photolabeling reaction. Of the various arylazido-beta-alanyl nucleotide derivatives studied, the NADP+ derivative influenced the enzymatic activity to the greatest extent; this is probably due to an ionic bond between enzyme and nucleotide, in addition to the covalent bond of the photolytic reaction.
D000345:Affinity Labels / Q000378:metabolism*; D001386:Azides; D001665:Binding Sites; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism*; D009243:NAD / Q000031:analogs & derivatives* / Q000378:metabolism; D010782:Photolysis; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013050:Spectrometry, Fluorescence
D000345:Affinity Labels; D001386:Azides; C020606:arylazido-beta-alanyl-NAD; D009243:NAD; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases