PHOTOAFFINITY-LABELING OF LACTATE-DEHYDROGENASE FROM PIG-HEART WITH ABIFUNCTIONAL NAD(-ANALOG())

zidophenylethyl)adenosine-P-2-4-(3-azidopyridinio) butyl diphosphate w as synthesized with an [8-C-14]adenine label. This bifunctional photoa ffinity labelling reagent inactivates lactate dehydrogenase from pig h eart upon irradiation with light of wavelength 300-180 nm. Stoichiomet ry of binding and enzymatic parameters suggest that the analogue is bo und to the coenzyme binding site and that adjacent residues are modifi ed, Four radioactive peptides were isolated by reverse-phase HPLC afte r tryptic digestion of the labelled protein. Amino-acid sequence analy sis identified the peptides and correlation with the three-dimensional structure of dogfish lactate dehydrogenase reveals that the peptides correspond to positions affecting the coenzyme binding site, consisten t with proper affinity labelling. Two of the peptides, Ile-77 --> Lys- 81 and Asp-82 --> Asn-88, are located close to the adenine binding sit e. Low recovery of Thr-86 in combination with the detection of additio nal products in the sequence analysis indicates that this residue is m odified by the photoaffinity label. The two other peptides (positions 119-124 and 318-328) are located next to the substrate binding site; t heir label is lost upon treatment with pyrophosphatase, showing that t hey are linked to the pyridinio moiety of the coenzyme analogue.