Glyoxalase I

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The glyoxalase system

Glyoxalase I (EC 4.4.1.5) is part of the glyoxalase system found in the cytosol of all cells. The glyoxalase system catalyzes the conversion of acyclic α-oxyaldehydes to α-hydroxyacid. The system is composed of two enzymes, glyoxalase I and glyoxalase II (EC 3.1.2.6), and a catalytic amount of GSH. Glyoxalase I is responsible for catalyzing the isomerization of hemithioacetals spontaneously formed by α-oxoaldehyde (RCOCHO) and GSH to produce S-2-hydroxyacylglutathione derivatives [RCH(OH)CO-SG]:

Glyoxalase II catalyzes the conversion of S-2-hydroxyacylglutathione derivatives into α-hydroxyacids and re-forms GSH. When glyoxalase I is inhibited in situ by a glyoxalase I inhibitor and by depletion GSH, the major physiological substrate of glyoxalase I, methylglyoxal, will accumulate significantly. Other substrates are hydroypyruvaldehyde (HOCH2COCHO), glyoxal (formed by lipid peroxidation and fragmentation of glycated proteins), and 4,5- doxovalerate (H-COCOCH2CH2CO2H). Glyoxalase I activity can effectively prevent the accumulation of these active α-oxyaldehydes, thereby inhibiting α-oxoaldehyde-mediated glycation reactions. Therefore, it is a key enzyme in the process of anti-glycation defense.  

Molecular properties of glyoxalase I

Glyoxalase I activity is present in all human tissues. On average, human tissues and blood cells contain 0.2 µg glyoxalase I per milligram of protein. The enzyme is a dimer and is expressed at a diallelic genetic locus, GLO. GLO encodes two similar subunits in heterozygotes. The three alloenzymes are named GLO 1-1, GLO 1-2 and GLO 2-2. The molecular mass of all the alloenzymes is 46 kDa (gel filtration), and the pI values of 4.8-5.1. But they have different charge densities and/or molecular shapes, so they can be resolved by ion-exchange chromatography and non-denaturing gel electrophoresis.

Each subunit of glyoxalase I contains a Zn²⁺ ion, but studies have found that glyoxalase I from E. coli is a Ni²⁺ metalloenzyme. So far, 61 glyoxalase I sequences have been reported, among which the glyoxalase I of humans, bacteria and plants exists in the form of dimers. The yeast enzymes of Saccharomyces cerevisiae and Schizosaccharomyces pombe are 32 kDa and 37 kDa monomers, respectively. The sequence identity between human glyoxalase I and the bacterial enzyme (Pseudomonas putida) is 55%, and with the yeast enzyme between residues 1-182 and 183-326 (Saccharomyces cerevisiae) is 47%, indicating that glyoxalase I of different origins may have evolved from a common ancestor.

Structure and catalytic mechanism of glyoxalase I

The three-dimensional structure of the complex of human glyoxalase I with S-benzylglutathione has been resolved, and each monomer is composed of two structurally equivalent domains. The active site exists at the dimer interface, the inhibitor and Zn²⁺ ion interact with the side chains from the two subunits. The glyoxalase I of Escherichia coli is composed of two identical subunits containing 135 amino acids. From the resolved crystal structure of the Ni²⁺-bound enzyme, it can be observed that each subunit consists of two domains, residues 3-60 and 72-126, that are linked by an intervening 12-residue segment.

Figure 1. Solid ribbon representations of the crystal structures of human and Escherichia coli glyoxalase I respectively (Thornalley, P. J. 2003)

The mechanism of the glyoxalase I reaction involves the transfer of base-catalyzed shielded-proton from C-1 to C-2 of the hemithioacetal, which binds to the active site to an ene-diol intermediate, and rapidly ketonization into a thioester product. It is currently believed that Glu-99 is the catalytic basis of the R-substrate enantiomer, and Glu-172 is the catalytic basis of the S-substrate enantiomer. Both reaction mechanisms form a cis-ene-diol intermediate (directly coordinated with Zn²⁺ ion): Glu-172 deprotonates it into cis-ene-diolate, and then reprotonates C-2 to form R-2- hydroxyacylglutathione product. By analogy with human enzymes, Glu-56 and Glu-122 may be bases involved in the catalytic mechanism.

Figure 2. Catalytic mechanism of human glyoxalase I for the isomerization of the R-hemithioacetal (Thornalley, P. J. 2003)