The Enzymatic Defence against Glycation in Health, Disease and Therapeutics 1343Glyoxalase I – structure, function and a criticalrole in the enzymatic defence against glycationP.J. Thornalley1Department of Biological Sciences, University of Essex, Central Campus, Wivenhoe Park, Colchester, Essex CO4 3SQ, U.K.AbstractGlyoxalase I is part of the glyoxalase system present in the cytosol of cells. The glyoxalase system catalysesthe conversion of reactive, acyclicα-oxoaldehydes into the corresponding α-hydroxyacids. Glyoxalase Icatalyses the isomerization of the hemithioacetal, formed spontaneously fromα-oxoaldehyde and GSH,to S-2-hydroxyacylglutathione derivatives [RCOCH(OH)-SG → RCH(OH)CO-SG], and in so doing decreases thesteady-state concentrations of physiologicalα-oxoaldehydes and associated glycation reactions. Physiolog-ical substrates of glyoxalase I are methylglyoxal, glyoxal and other acyclicα-oxoaldehydes. Humanglyoxalase I is a dimeric Zn2+metalloenzyme of molecular mass 42 kDa. Glyoxalase I from Escherichia coli isaNi2+metalloenzyme. The crystal structures of human and E. coli glyoxalase I have been determined to 1.7and 1.5 Å resolution. The Zn2+site comprises two structurally equivalent residues from each domain – Gln-33A, Glu-99A, His-126B, Glu-172B and two water molecules. The Ni2+binding site comprises His-5A, Glu-56A,His-74B, Glu-122B and two water molecules. The catalytic reaction involves base-catalysed shielded-protontransfer from C-1 to C-2 of the hemithioacetal to form an ene-diol intermediate and rapid ketonization to thethioester product. R-andS-enantiomers of the hemithioacetal are bound in the active site, displacingthe water molecules in the metal ion primary co-ordination shell. It has been proposed that Glu-172 is thecatalytic base for the S-substrate enantiomer and Glu-99 the catalytic base for the R-substrate enantiomer;Glu-172 then reprotonates the ene-diol stereospecifically to form the R-2-hydroxyacylglutathione product.By analogy with the human enzyme, Glu-56 and Glu-122 may be the b ases involved in the catalyticmechanism of E. coli glyoxalase I. The suppression ofα-oxoaldehyde-mediated glycation by glyoxalaseI is particularly important in diabetes and uraemia, whereα-oxoaldehyde concentrations are increased.Decreased glyoxalase I activity in situ due to the aging process and oxidative stress results in increasedglycation and tissue damage. Inhibition of glyoxalase I pharmacologically with specific inhibitors leads to theaccumulation ofα-oxoaldehydes to cytotoxic levels; cell-permeable glyoxalase I inhibitors are antitumourand antimalarial agents. Glyoxalase I has a critical role in the prevention of glycation reactions mediated bymethylglyoxal, glyoxal and otherα-oxoaldehydes in vivo.The glyoxalase systemGlyoxalase I (EC 4.4.1.5) is part of the glyoxalase systempresent in the cytosol of all cells. The glyoxalase system cata-lyses the conversion of reactive, acyclic α-oxoaldehydesinto the corresponding α-hydroxyacids. It is composedof two enzymes, glyoxalase I and glyoxalase II (EC3.1.2.6), and a catalytic amount of GSH. Glyoxalase Icatalyses the isomerization of the hemithioacetal, formedspontaneously from α-oxoaldehyde (RCOCHO) andGSH, into S-2-hydroxyacylglutathione derivativesKey words: glutathione, glycation, glyoxalase, methylglyoxal, oxidative stress.Abbreviations used: AGE, advanced glycation end-product; CDNB, 1-chloro-2,4-dinitrobenzene;SpBrBzGSH, S-p-bromobenzylglutathione; SpBrBzGSHCp2, S-p-bromobenzylglutathione cyclo-pentyl diester; SpBrBzGSHEt2, S-p-bromobenzylglutathione ethyl diester; CEdG, N2-(1-carboxy-ethyl)deoxyguanosine; CEL, Nε-(1-carboxyethyl)lysine; dG-MG, 6,7-dihydro-6,7-dihydroxy-6-methylimidazo-[2,3-b]purine-9(8)one; dG-MG2, N2,7-bis-(1-hydroxy-2-oxopropyl)deoxy-guanosine; MG-H1, Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl)ornithine; MOLD, methylglyoxal-derived lysine dimer [1,3-di(Nε-lysino)-4-methyl-imidazolium salt].1e-mail [email protected][RCH(OH)CO-SG]:RCOCHO + GSH ↔ RCOCH(OH)-SG→ RCH(OH)CO-SGFor the methylglyoxal–glutathione hemithioacetal andhuman glyoxalase I, the Kmis 71–130 µMandthekcatis(7–11) × 104min−1. Glyoxalase II catalyses the conversion ofS-2-hydroxyacylglutathione derivatives into α-hydroxyacidsand re-forms GSH consumed in the glyoxalase I-catalysedreaction step. The major physiological substrate for glyox-alase I is methylglyoxal, and this accumulates markedly whenglyoxalase I is inhibited in situ by cell-permeable glyoxalaseI inhibitors and by depletion of GSH [1–3]. Methylglyoxalis formed mainly by the degradation of triose phosphates,and also by the metabolism of ketone bodies, threoninedegradation and the fragmentation of glycated proteins.Other substrates are glyoxal (formed by lipid peroxidationand the fragmentation of glycated proteins), hydroypyr-uvaldehyde (HOCH2COCHO) and 4,5-doxovalerateC 2003 Biochemical Society1344 Biochemical Society Transactions (2003) Volume 31, part 6(H-COCOCH2CH2CO2H) [1,4]. Glyoxalase I activityprevents the accumulation of these reactive α-oxoaldehydesand thereby suppresses α-oxoaldehyde-mediated glycationreactions [5]. It is, therefore, a key enzyme of the anti-glycation defence.Molecular properties of glyoxalase IGlyoxalase I activity is present in all human tissues. Specificactivities of fetal tissues are approx. 3 times higher than thoseof corresponding adult tissues. There is approx. 0.2 µgofglyoxalase I per mg of protein in human tissues and bloodcells. Human glyoxalase I is a dimer, expressed at a diallelic ge-netic locus, GLO, which encodes two similar subunits in het-erozygotes; the three alloenzymes are designated GLO 1-1,GLO 1-2 and GLO 2-2. All alloenzymes have molecularmass of 46 kDa (gel filtration) or 42 kDa (sequence), andpI values of 4.8–5.1; however, they have distinctive chargedensities and/or molecular shapes and are resolved by ionexchange chromatography and non-denaturing gel elec-trophoresis. Each subunit contains one Zn2+ion [1].Surprisingly, glyoxalase I from Escherichia coli is a Ni2+metalloenzyme [6].To date, 61 glyoxalase I sequences have been reported,from human, mouse, yeast (Saccharomyces cerevisiae andSchizosaccharomyces pombe), plants (Arabidopsis thaliana,Avicennia marina, Brassica oleracea, Brassica juncea, Cicerarietinum, Glycine max, Oryza sativa, Sporobolus stapfi-anus, Solanum lycopersicu and Triticum aestivum), insects(Drosophila melanogaster and Anopheles gambiae),