Carboxyethyllysine in a protein: native carbonyl reductase/NADP(+)-dependent prostaglandin dehydrogenase.

Abstract

Two different forms of the monomeric NADP+-ilnked prostaglandin dehydrogenase/carbonyl reductase were purified from human placenta and shown to differ by the m ation of a lysine residue. The modified and the unmodified proteins were reproducibly recovered In a ratio of -1:3, and both were chemically stable. The modifed form was more acidic (pI 7.4 versus pI 7.7) but I i habl from the unmoded form in specificity and activity. Amino acid analysis, sequence analysis, mass spectrometry, and chemical synthesis identified the modified residue as N6-(lcarboxyethyl)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 assiment to short-chain dehydrogenases. Alignments and model-building into the tertiary structure of 3a/20j3-hydroxysteroid dehydrogenase show that carbonyl reductase has an extra loop (positions 149-189) that forms a separate extension and replaces a backbone C-terminal ,3-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.The biological inactivation of prostaglandins is mediated by 15-hydroxyprostaglandin dehydrogenase. Two different types of this cytosolic activity, the most active one using NAD+ and the other using NADP+, were detected early in a number of tissues (1-3). We have characterized the NAD+-linked enzyme as a short-chain dehydrogenase (4), with properties typical of that enzyme family (5). A crystallographic structure of the protein 3a/20,B-hydroxysteroid dehydrogenase, which is within this family, has recently become available (6), as has that of another, distantly related member of the family, dihydropteridine reductase, which is strikingly similar, except for some loop and turn regions (7). These and other short-chain dehydrogenases contain a functionally important tyrosine residue (4, 8-10) and a segment sensitive to proteolysis (10).The NADP+ enzyme type also acts on the 9-keto group of prostaglandins in the reverse direction (3, 11, 12), thus constituting a prostaglandin 9-keto reductase and can then convert prostaglandin E2 to prostaglandin F2<t. However, quinones are far better substrates than prostaglandins, and the possibility that carbonyl reductase and NADP+-dependent prostaglandin dehydrogenase are closely related or even identical was suggested early (13-15) and has recently been substantiated (16). Consequently, the nomenclature of this NADPH-linked protein is complicated, and the enzyme has been given no less than three EC numbers (EC 1. MATERIALS AND METHODS Enzyme Purification. S...

Identification of the reactive cysteine residue (Cys227) in human carbonyl reductase

“…The functional role of the reactive cysteine, however, remains speculative. Based on the known tertiary structure of the bacterial 3a/20b-hydroxysteroid dehydrogenase [9], Jo Èrnvall and colleagues have constructed a three-dimensional model of human carbonyl reductase [25]. Although the model has some uncertainties, particularly in the C-terminal part of the peptide chain, it clearly identifies the residues constituting the a-helices and b-pleated sheets of the Rossmann fold and positions Cys227 in the C-terminal part of the bF strand, where it precedes a highly conserved proline residue.…”

Mass spectrometric analysis of rat ovary and testis cytosolic glutathione S-transferases (GSTs): identification of a novel class-Alpha GST, rGSTA6*, in rat testis

“…Assuming that these proteins have equivalent ionization efficiency, they were present in the proportions 7 : 10 : 4 in our sample. The human carbonyl reductase is N-terminally acetylated and has a carboxyethyl-lysine at position 238 [36]. The molecular mass of the modified human enzyme is 114 Da higher than that predicted from the cDNA sequence.…”

Protein Glycation, Diabetes, and Aging

“…AGES in which a I-carboxyalkyl group is attached to a free amino group of an amino acid residue, such as NE-(carboxymethyl)lysine (CML) and NE-(l-carboxyethyl)lysine (CEL), are found in proteins and in free form in vivo (Liardon et al, 1987;Krook et al, 1993;Ahmed et al, 1997). They may arise either from reaction of lysine residues with glyoxal derivatives (Glomb and Monnier, 1995) or from autoxidation of early-stage AGES such as the Amadori product (Brinkmann Frye et al, 1998).…”