Role of Lysine‐183 in d ‐Glyceraldehyde‐3‐Phosphate Dehydrogenases

The acetylation of the ɛNH 2 of lysine‐183 of rabbit muscle, sturgeon muscle and yeast glyceral‐dehyde‐3‐phosphate dehydrogenases at pH 8.5, by S→N acetyl transfer follows a first‐order process. Essentially, four lysine residues are modified per enzyme tetramer. When enzymic assays are carried out under saturating conditions of the three substrates (NAD + , glyceraldehyde 3‐phosphate and arsenate), the N ‐acetylated rabbit and sturgeon muscle enzymes show values of V which are 90% and 80% respectively of that of the native enzymes, while for the N ‐acetylated yeast enzyme it is only 20%. The K m values for the three substrates are not significantly affected by the N ‐acetylation of the two muscle enzymes. However, the N ‐acetylated yeast enzyme yields an increased K m for NAD + only. By spectrophotometric titration at the Racker band, it was shown that each of the three N ‐acetylated enzymes still binds four NAD + per tetramer. Spectrofluorimetric titration of the binding of NAD + to the N ‐acetylated rabbit muscle enzyme revealed that acetylation of lysine‐183 affected essentially the first three macroscopic NAD + dissociation constants. The fourth macroscopic dissociation constant remained of the same order of magnitude as that found for the native enzyme. However, the calculated microscopic dissociation constants show that the affinities of the second, third and fourth NAD + are of the same order. This result indicates that acetylation of lysine‐183 desensitized the anticooperative binding process of the coenzyme. Furthermore, it was observed that this chemical modification also affected the profile of arsenate activation for the three enzymes. Spectroscopic, ultracentrifugation and thermostability studies failed to show any significant conformational change due to the N ‐acetylation. In conclusion, our results show that the four lysine‐183 residues per tetramer of muscle or yeast glyceraldehyde‐3‐phosphate dehydrogenase are equally accessible to N ‐acetylation. Reaction with the yeast enzyme is much slower than that with the muscle enzymes. It is shown further that lysine‐183 is implicated neither in the catalytic site nor in the substrate binding site. However, this residue seems to play an important role in the area of contact between the two adjacent subunits through which flows conformational information incidental to the binding of NAD + or arsenate to the protein. This result is in agreement with X‐ray crystallographic studies.