br Conclusion br Introduction The intracellularly generated

Conclusion
Introduction The intracellularly generated metabolite methylglyoxal (MG, 2-oxopropanal) acts as a potent electrophile causing irreparable cellular damage if allowed to build to cytotoxic concentrations [1], [2], [3], [4], [5], [6]. The glyoxalase (Glx) system is an enzyme couple critical for the detoxification of MG. The system consists of two enzymes, glyoxalase I (GlxI, S-d-lactoylglutathione lyase; EC 4.4.1.5) and glyoxalase II (GlxII, S-2-hydroxyacylglutathione hydrolase; EC 3.1.2.6) (Fig. 1) [7]. The first enzyme, GlxI, converts the hemithioacetal, a product formed by the non-enzymatic reaction of MG and a thiol cofactor/cosubstrate such as glutathione (GSH; γ-Glu-Cys-Gly), to S-d-lactolyglutathione. This thioester is then hydrolyzed by the second enzyme, GlxII, to produce a non-toxic 2-hydroxycarboxylic vps34 in the form of d-lactate and the regenerated thiol cofactor. There is evidence that the intermediate, S-d-lactoylglutathione, can control intracellular pH in bacteria through interaction with the KefB potassium efflux system [8]. There are several different thiol cofactors found in various organisms. GSH is the most prominent intracellular thiol in eukaryotes and some prokaryotes [9], [10], [11], [12]. This thiol is involved in many biological functions including aromatic metabolism, maintenance of cellular redox potentials and several detoxification mechanisms [11], [13]. In E. coli, a novel GSH and spermidine (spd) conjugate was discovered and designated as glutathionylspermidine (GspdSH, Fig. 1). This complex glutathione and its disulfide form (glutathionylspermidine disulfide, (GspdS)2), under anaerobic conditions or stationary phase, compose approximately 80% of all produced GSH in E. coli[14], [15]. An elevated level of GspdSH production is found to be in proportion to bacterial cell density [14], [15], while the amount of (GspdS)2 is decreased when cellular stress conditions prevail. GspdSH is also recognized to act more efficiently toward protecting cells against radical or oxidant-induced damage than its disulfide form [14], [16]. Analysis of these experimental results suggests that GspdSH could be a reasonable alternate cofactor for the Glx system under these conditions. Interestingly, the glyoxalase enzymes are also conserved in organisms that produce major intracellular thiols other than GSH. Recent studies on Leishmania and Trypanosoma have indicated that their corresponding Glx systems have evolved to use the bis-glutathionyl derivative of spermidine, trypanothione (T(SH)2, N1,N8-bisglutathionylspermidine) (Fig. 1), the predominant thiol of these organisms, as a cofactor [17], [18], [19], [20], [21]. The Glx system has been investigated with respect to mechanistic, structural and metabolic aspects. GlxI enzymes are metalloenzymes that can be divided into two classes according to their metal activation profile, Zn-activation (i.e., H. sapiens GlxI [22]) or Ni/Co-activation (non-Zn-activated i.e., E. coli GlxI [23]). The active metallated forms of these enzymes require a metal environment that is octahedral in nature, with four protein residues serving as metal ligands, along with two water molecules completing the geometry [24], [25], [26]. This occurs for the Zn-bound active human GlxI as well as the catalytically active Ni-bound E. coli GlxI. However, the inactive Zn-bound form of the E. coli GlxI approximates a trigonal bipyramidal geometry around the Zn ion with only one water molecule present [26]. Mechanistically it is suggested, based on analysis of the X-ray structures of inhibitors bound to the H. sapiens GlxI, that the arrival of the hemithioacetal substrate at the catalytic site results in the replacement of the two active site water ligands by the two oxygens from the hemithioacetal substrate. This is likely followed by a series of deprotonation, reprotonation steps (Fig. 1), resulting in thioester product formation [24], [25]. This mechanism is supported by reported solvent isotope incorporation studies as well as observations of a kinetic isotope effect using deuterated α-ketoaldehydes with yeast GlxI (Zn-activated enzyme) [27], [28]. To date, no studies have been reported that investigate the Ni-activation class of GlxI in this manner.