Biochem. J. (2013) 453, 1–15 (Printed in Great Britain) doi:10.1042/BJ20121743 1 REVIEW ARTICLE The glyoxalase pathway: the first hundred years ... and beyond Marta SOUSA SILVA 1 , Ricardo A. GOMES 1 , Antonio E. N. FERREIRA, Ana PONCES FREIRE and Carlos CORDEIRO 2 Centro de Qu´ ımica e Bioqu´ ımica, Departamento de Qu´ ımica e Bioqu´ ımica, Faculdade de Ciˆ encias da Universidade de Lisboa, Portugal The discovery of the enzymatic formation of lactic acid from methylglyoxal dates back to 1913 and was believed to be associated with one enzyme termed ketonaldehydemutase or glyoxalase, the latter designation prevailed. However, in 1951 it was shown that two enzymes were needed and that glutathione was the required catalytic co-factor. The concept of a metabolic pathway defined by two enzymes emerged at this time. Its association to detoxification and anti-glycation defence are its presently accepted roles, since methylglyoxal exerts irreversible effects on protein structure and function, associated with misfolding. This functional defence role has been the rationale behind the possible use of the glyoxalase pathway as a therapeutic target, since its inhibition might lead to an increased methylglyoxal concentration and cellular damage. However, metabolic pathway analysis showed that glyoxalase effects on methylglyoxal concentration are likely to be negligible and several organisms, from mammals to yeast and protozoan parasites, show no phenotype in the absence of one or both glyoxalase enzymes. The aim of the present review is to show the evolution of thought regarding the glyoxalase pathway since its discovery 100 years ago, the current knowledge on the glyoxalase enzymes and their recognized role in the control of glycation processes. Key words: glycation, glyoxalase, Maillard reaction, methylgly- oxal. IN THE BEGINNING The observation that yeast cell-free extracts could catalyse the formation of ethanol and carbon dioxide from glucose heralded modern biochemistry. At the time, little was known of the chemical composition of living cells and much less so regarding the chemical reactions that enable life. Attempts to reproduce biochemical processes revealed that a few molecules might be intermediates in the process of fermentation. One such molecule was methylglyoxal, readily formed under alkaline conditions from glyceraldehyde or dihydroxyacetone. The discovery in 1913 of an enzymatic activity that converted methylglyoxal into lactic acid appeared to show the path from glucose to pyruvic acid or ethanol [1,2]. Initially termed ketonaldehydemutase, it was later that the designation of GLO (glyoxalase) became accepted. Methylglyoxal and GLO became a focal point in glycolysis. At that time, the accepted model for glycolysis consisted of a splitting of glucose into two trioses that form methylglyoxal which was then converted into lactate through the action of GLO [3]. Although this scheme held its own for a long time, several observations dismissed a glycolytic role for methylglyoxal and GLO. Indeed, it was soon recognized that GLO required glutathione as a co-factor, whereas glutathione has no effect on glycolysis [4,5]. The search for an enzymatic origin of methylglyoxal was hampered by the discovery that DHAP (dihydroxyacetone phosphate) and GAP (D-glyceraldehyde 3-phosphate) are chem- ically unstable in physiological conditions and produce methyl- glyoxal non-enzymatically [6]. Methylglyoxal was therefore assumed not to be a key metabolite, entering the realm of chemical artefacts. In 1948 a puzzling observation was made by Hopkins and Morgan [7] of an isolated factor that increased the rate of the GLO-catalysed reaction. This observation was soon rationalized as indicating the presence of a second enzyme by Crook and Law in 1950 [8], although the proof that two enzymes were required to produce lactate from methylglyoxal came later from Racker [9] with subsequent confirmation by Crook and Law [10]. Racker [9] showed that GLO activity could be accounted for by the action of two enzymes that he partially purified and named GLO1 and GLO2. He also showed the existence of an intermediate with specific spectral characteristics and demonstrated that both GLOs catalysed virtually irreversible reactions (Figure 1). This was one of the most relevant works in the field, being one of the most fruitful demonstrations of the power of spectrophotometric methods in enzymology. It was also the basis of the first specific enzymatic assay for glutathione. Later, the lactic acid formed through the action of the GLO pathway was found to be D-lactate, ruling out methylglyoxal and GLOs from glycolysis [11]. This raised the question of the physiological role of the GLO pathway since this two-enzyme system had no physiological substrate and led to a dead-end product. In the late sixties, Szent-Gyorgyi [12,13] proposed an electronic theory of cancer on the basis of the regulation of the conducting properties of proteins as evidenced by quantum biochemistry studies. A reversible reaction with dicarbonyls would lead to a change in protein conductivity. Since glutathione and GLO activity could be related to cell proliferation, a retine–promine Abbreviations used: AGE, advanced glycation end-product; ATTR, transthyretin amyloidosis; CEdG, N 2 -(1-carboxyethyl)-deoxyguanosine; CEL, N ε - (carboxyethyl)lysine; CML, N-(carboxymethyl)lysine; DHAP, dihydroxyacetone phosphate; GAP, D-glyceraldehyde 3-phosphate; GdG, 3-(2 ′ -deoxyribosyl)- 6,7-dihydro-6,7-dihydroxyimidazo[2,3-b]purin-9(8)one; GLO, glyoxalase; Hsp, heat-shock protein; LDL, low-density lipoprotein; MAGE, methylglyoxal- derived AGE; MGdG, 3-(2 ′ -deoxyribosyl)-6,7-dihydro-6,7-dihydroxy-6-methylimidazo-[2,3-b]purine-9(8)one; MOLD, methylglyoxal–lysine dimer; 8-OxodG, 8-hydroxydeoxyguanosine; TIM, triose phosphate isomerase. 1 These authors contributed equally to this work. 2 To whom correspondence should be addressed (email cacordeiro@fc.ul.pt). c  The Authors Journal compilation c  2013 Biochemical Society