CARBOXYETHYLLYSINE IN A PROTEIN - NATIVE CARBONYL REDUCTASE/NADP+-DEPENDENT PROSTAGLANDIN DEHYDROGENASE

Two different forms of the monomeric NADP+-linked prostaglandin dehydrogenase/carbonyl reductase were purified from human placenta and shown to differ by the modification of a lysine residue. The modified and the unmodified proteins were reproducibly recovered in a ratio of almost-equal-to 1:3, and both were chemically stable. The modified form was more acidic (pI almost-equal-to 7.4 versus pI almost-equal-to 7.7) but indistinguishable from the unmodified form in specificity and activity. Amino acid analysis, sequence analysis, mass spectrometry, and chemical synthesis identified the modified residue as N6-(1-carboxyethyl)lysine with C-2 of propionic acid attached to the side-chain N of Lys-238. This compound can be formed from the lysine residue and pyruvate via a Schiff base and subsequent reduction. The enzyme and its NAD+-dependent counterpart are distantly related (23% residue identity) and have the same family assignment to short-chain dehydrogenases. Alignments and model-building into the tertiary structure of 3alpha/20beta-hydroxysteroid dehydrogenase show that carbonyl reductase has an extra loop (positions 149-189) that forms a separate extension and replaces a backbone C-terminal beta-strand. This change affects the substrate pocket, explaining the different substrate specificities but conserves residues of known functional importance. Carboxyethyllysine at position 238 corresponds to a proteolysis-sensitive position in several short-chain dehydrogenases, less well-defined in the model but close to a surface, and is compatible with the accessibility and enzyme properties observed.