Identification of Lysines Reactive with Pyridoxal 5′‐Phosphate in Glyceraldehyde‐3‐phosphate Dehydrogenase

Rabbit muscle glyceraldehyde‐3‐phosphate dehydrogenase is almost completely inhibited by reaction of 4 moles of pyridoxal 5′‐phosphate per mole of subunit chain of holoenzyme. Reduction of the pyridoxal 5′‐phosphate inactivated holoenzyme with NaBH 4 followed by carboxymethylation of thiol groups, and subsequent tryptic digestion, allowed the separation of two strongly fluorescent peptides. These have been identified as peptides T19+T20 (residues 184 to 192) and peptides T22+T23 (residues 198 to 216) of the sequence published by Harris and Perham for porcine enzyme, in which lysines 191 and 212 are blocked by pyridoxal 5′‐phosphate. Evidence for other lysyl residues reacting to a lesser extent with the reagent is reported: among such residues, only lysine‐2(contained in the N‐terminal peptide) has been unambigously identified. No reaction takes place at lysine‐183. The homology between the sequence around lysine‐212 and the sequence around the essential lysine residue in glutamate dehydrogenase is stressed. The role of pyridoxal 5′‐phosphate reactive lysines in the mechanism of glyceraldehyde‐3‐phosphate dehydrogenase is discussed.