Lactoylglutathione lyase

In is_associated_with::enzymology, a lactoylglutathione lyase  (also known as glyoxalase I) is an is_associated_with::enzyme that catalyzes the is_associated_with::isomerization of hemithioacetal adducts, which are formed in a spontaneous reaction between a glutathionyl group and is_associated_with::aldehydes such as is_associated_with::methylglyoxal.


 * glutathione + methylglyoxal $$\rightleftharpoons$$ hemithioacetal adduct $$\rightleftharpoons$$ (R)-S-lactoylglutathione

Glyoxalase I derives its name from its catalysis of the first step in the is_associated_with::glyoxalase system, a critical two-step detoxification system for is_associated_with::methylglyoxal. Methylglyoxal is produced naturally as a byproduct of normal biochemistry, but is highly toxic, due to its chemical reactions with is_associated_with::proteins, is_associated_with::nucleic acids, and other cellular components. The second detoxification step, in which (R)-S-lactoylglutathione is split into glutathione and D-lactate, is carried out by is_associated_with::glyoxalase II, a is_associated_with::hydrolase. Unusually, these reactions carried out by the glyoxalase system does not oxidize glutathione, which usually acts as a is_associated_with::redox is_associated_with::coenzyme. Although is_associated_with::aldose reductase can also detoxify methylgloxal, the glyoxalase system is more efficient and seems to be the most important of these pathways. Glyoxalase I is an attractive target for the development of drugs to treat infections by some parasitic protozoa, and is_associated_with::cancer. Several inhibitors of glyoxalase I have been identified, such as S-(N-hydroxy-N-methylcarbamoyl)glutathione.

Glyoxalase I is classified as a carbon-sulfur is_associated_with::lyase although, strictly speaking, the enzyme does not form or break a carbon-sulfur bond. Rather, the enzyme shifts two hydrogen atoms from one carbon atom of the methylglyoxal to the adjacent carbon atom. In effect, the reaction is an intramolecular is_associated_with::redox reaction; one carbon is oxidized whereas the other is reduced. The mechanism proceeds by subtracting and then adding is_associated_with::protons, forming an enediolate intermediate, rather than by transferring is_associated_with::hydrides. Unusually for a is_associated_with::metalloprotein, this enzyme shows activity with several different metals. Glyoxalase I is also unusual in that it is stereospecific in the second half of its mechanism, but not in the first half. Structurally, the enzyme is a domain-swapped dimer in many species, although the two subunits have merged into a monomer in is_associated_with::yeast, through is_associated_with::gene duplication.

Nomenclature
The systematic name of this enzyme class is (R)-S-lactoylglutathione methylglyoxal-lyase (isomerizing glutathione-forming); other names include methylglyoxalase, aldoketomutase, ketone-aldehyde mutase, and (R)-S-lactoylglutathione methylglyoxal-lyase (isomerizing). In some instances, the glutathionyl moiety may be supplied by is_associated_with::trypanothione, the analog of glutathione in parasitic protozoa such as the trypanosomes. The human gene for this enzyme is called is_associated_with::GLO1.

Gene
Lactoylglutathione lyase in humans is encoded by the GLO1 is_associated_with::gene.

Structure
Several structures of glyoxalase I have been solved. Four structures of the human form have been published, with PDB accession codes, , , and. Five structures of the is_associated_with::Escherichia coli form have been published, with accession codes, , , , and. Finally, one structure of the trypanothione-specific version from is_associated_with::Leishmania major has been solved,. In all these cases, the is_associated_with::quaternary structure of the biological unit is a domain-swapped dimer, in which the active site and the 8-stranded is_associated_with::beta sheet is_associated_with::secondary structure is formed from both subunits. However, in is_associated_with::yeast such as is_associated_with::Saccharomyces cerevisiae, the two subunits have fused into a single monomer of double size, through is_associated_with::gene duplication. Each half of the structural dimer is a sandwich of 3-4 alpha helices on both sides of an 8-stranded antiparallel beta sheet; the dimer interface is largely composed of the face-to-face meeting of the two beta sheets.

The tertiary and quaternary structures of glyoxalase I is similar to those of several other types of proteins. For example, glyoxalase I resembles several proteins that allow bacteria to resist antibiotics such as is_associated_with::fosfomycin, is_associated_with::bleomycin and is_associated_with::mitomycin. Likewise, the unrelated enzymes is_associated_with::methylmalonyl-CoA epimerase, is_associated_with::3-demethylubiquinone-9 3-O-methyltransferase and numerous is_associated_with::dioxygenases such as is_associated_with::biphenyl-2,3-diol 1,2-dioxygenase, is_associated_with::catechol 2,3-dioxygenase, is_associated_with::3,4-dihydroxyphenylacetate 2,3-dioxygenase and is_associated_with::4-hydroxyphenylpyruvate dioxygenase all resemble glyoxalase I in structure. Finally, many proteins of unknown or uncertain function likewise resemble glyoxalase I, such as At5g48480 from the plant, is_associated_with::Arabidopsis thaliana.

The active site has four major regions.

Function


The principal physiological function of glyoxalase I is the detoxification of is_associated_with::methylglyoxal, a reactive 2-oxoaldehyde that is cytostatic at low concentrations and cytotoxic at millimolar concentrations. Methylglyoxal is a by-product of normal biochemistry that is a carcinogen, a mutagen and can chemically damage several components of the cell, such as proteins and nucleic acids. Methylglyoxal is formed spontaneously from dihydroxyacetone phosphate, enzymatically by triosephosphate isomerase and methylglyoxal synthase, as also in the catabolism of is_associated_with::threonine.

To minimize the amount of toxic methylglyoxal and other reactive 2-oxoaldehydes, the is_associated_with::glyoxalase system has evolved. The methylglyoxal reacts spontaneously with reduced is_associated_with::glutathione (or its equivalent, is_associated_with::trypanothione), forming a hemithioacetal. The glyoxalase system converts such compounds into D-lactate and restored the glutathione. In this conversion, the two carbonyl carbons of the 2-oxoaldehyde are oxidized and reduced, respectively, the aldehyde being oxidized to a carboxylic acid and the acetal group being reduced to an alcohol. The glyoxalase system evolved very early in life's history and is found nearly universally through life-forms.

The glyoaxalase system consists of two enzymes, glyoxalase I and is_associated_with::glyoxalase II. The former enzyme, described here, rearranges the hemithioacetal formed naturally by the attack of is_associated_with::glutathione on methylglyoxal into the product. Glyoxalase II hydrolyzes the product to re-form the glutathione and produce D-lactate. Thus, glutathione acts unusually as a is_associated_with::coenzyme and is required only in catalytic (i.e., very small) amounts; normally, glutathione acts instead as a is_associated_with::redox couple in oxidation-reduction reactions.

The glyoxalase system has also been suggested to play a role in regulating cell growth and in assembling is_associated_with::microtubules.

Properties
Glyoxalase I requires bound metal ions for catalysis. The human enzyme and its counterparts in yeast (is_associated_with::Saccharomyces cerevisiae) and is_associated_with::Pseudomonas putida use divalent is_associated_with::zinc, Zn2+. By contrast, the prokaryotic versions often use a is_associated_with::nickel ion. Interestingly, the glyoxalase I found in eukaryotic trypanosomal parasites such as is_associated_with::Leishmania major and is_associated_with::Trypanosoma cruzi can also use nickel for activity, possibly reflecting an acquisition of their GLO1 gene by is_associated_with::horizontal gene transfer.

A striking property of glyoxalase I is its lack of specificity for the catalytic metal ion. Most enzymes prefer to bind one particular type of metal, and their catalytic activity depends on having bound that metal. For example, is_associated_with::oxidoreductases often use a specific is_associated_with::metal ion such as is_associated_with::iron, is_associated_with::manganese or is_associated_with::copper and will fail to function if their preferred metal ion is replaced, due to differences in the redox potential; thus, the ferrous is_associated_with::superoxide dismutase cannot function if its catalytic iron is replaced by manganese, and vice versa. By contrast, although human glyoxalase I prefers to use divalent zinc, it is able to function with many other divalent metals, including is_associated_with::magnesium, is_associated_with::manganese, is_associated_with::cobalt, is_associated_with::nickel and even is_associated_with::calcium.; however, the enzyme is inactive with the ferrous cation. Similarly, although the prokaryotic glyoxalase I prefers nickel, it is able to function with cobalt, manganese and is_associated_with::cadmium; however, the enzyme is inert with bound zinc, due to a change in is_associated_with::coordination geometry from octahedral to trigonal bipyramidal. Structural and computational studies have revealed that the metal binds the two carbonyl oxygens of the methylglyoxal moiety at two of its coordination sites, stabilizing the enediolate anion intermediate.

Another unusual property of glyoxalase I is its inconsistent stereospecificity. The first step of its reaction mechanism (the abstraction of the proton from C1 and subsequent protonation of O2) is not stereospecific, and works equally well regardless of the initial chirality at C1 in the hemithioacetal substrate. The resulting enediolate intermediate is achiral, but the second step of the reaction mechanism (the abstraction of a proton from O1 and subsequent protonation of C2) is definitely stereospecific, producing only the (S) form of D-lactoylglutathione. This is believed to result from the two is_associated_with::glutamates bound oppositely on the metal ion; either one is able to carry out the first step, but only one is able to carry out the second step. The reason from this asymmetry is not yet fully determined.

Mechanism
The is_associated_with::methylglyoxal molecule consists of two is_associated_with::carbonyl groups flanked by a is_associated_with::hydrogen atom and a is_associated_with::methyl group. In the discussion below, these two carbonyl carbons will be denoted as C1 and C2, respectively. In both the hemithioacetal substrate and the (R)-S-lactoylglutathione product, the is_associated_with::glutathione moiety is bonded to the C1 carbonyl group.

The basic mechanism of glyoxalase I is as follows. The substrate hemithioacetal is formed when a molecule of is_associated_with::glutathione — probably in its reactive is_associated_with::thiolate form — attacks the C1 carbonyl of methylglyoxal or a related compound, rendering that carbon tetravalent. This reaction occurs spontaneously in the cell, without the involvement of the enzyme. This hemithioacetal is then bound by the enzyme, which shifts a hydrogen from C1 to C2. The C2 carbonyl is reduced to a tetravalent alcohol form by the addition of two protons, whereas the C1 carbonyl is restored by losing a hydrogen while retaining its bond to the glutathione moiety.

A computational study, combined with the available experimental data, suggests the following atomic-resolution mechanism for glyoxalase I. In the active site, the catalytic metal adopts an octahedral coordination geometry and, in the absence of substrate, binds two waters, two opposite is_associated_with::glutamates, a is_associated_with::histidine and one other sidechain, usually another histidine or is_associated_with::glutamates. When the substrate enters the active site, the two waters are shed and the two carbonyl oxygens of the substrate are bound directly to the metal ion. The two opposing glutamates add and subtract protons from C1 and C2 and their respective oxygens, O1 and O2. The first half of the reaction transfers a proton from C1 to O2, whereas the second half transfers a proton from O1 to C2. The former reaction may be carried out by either of the opposing glutamates, depending on the initial chirality of C1 in the hemithioacetal substrate; however, the second half is stereospecific and is carried out by only one of the opposing glutamates.

Proton vs. hydride transfer
Glyoxalase I was originally believed to operate by the transfer of a is_associated_with::hydride, which is a is_associated_with::proton surrounded by two is_associated_with::electrons (H–). In this, it was thought to resemble the classic is_associated_with::Cannizzaro reaction mechanism, in which the attack of a hydroxylate on an aldehyde renders it into a tetravalent alcohol anion; this anion donates its hydrogens to a second aldehyde, forming a carboxylic acid and an alcohol. (In effect, two identical aldehydes reduce and oxidize each other, leaving the net oxidation state the same.)

In glyoxalase I, such a hydride-transfer mechanism would work as follows. The attack of the glutathione would leave a charged O– and the aldehyde hydrogen bound to C1. If the carbonyl oxygen of C2 can secure a hydrogen from an obliging acidic sidechain of the enzyme, forming an alcohol, then the hydrogen of C1 might simultaneously slide over with its electrons onto C2 (the hydride transfer). At the same time, the extra electron on the oxygen of C1 could reform the double bond of the carbonyl, thus giving the final product.

An alternative (and ultimately correct) mechanism using is_associated_with::proton (H+) transfer was put forward in the 1970s. In this mechanism, a basic sidechain of the enzyme abstracts the aldehyde proton from C1; at the same time, the a proton is added to the oxygen of C2, thus forming a enediol. The ene means that a double bond has formed between C2 and C1, from the electrons left behind by the abstraction of the aldehyde proton; the diol refers to the fact that two alcohols have been made of the initial two carbonyl groups. In this mechanism, the intermediate forms the product by adding another proton to C2.

It was expected that solvent protons would contribute to forming the product from the enediol intermediate of the proton-transfer mechanism and when such contributions were not observed in tritiated water, 3H1O, the hydride-transfer mechanism was favored. However, an alternate hypothesis — that the enzyme active site was deeply buried away from water — could not be ruled out and ultimately proved to be correct. The first indications came when ever-increasing temperatures showed ever-increasing incorporation of tritium, which is consistent with proton transfer and unexpected by hydride transfer. The clinching evidence can with studies of the hydrogen-deuterium is_associated_with::isotope effect on substrates fluorinated on the methyl group and deuterated on the aldehyde. The fluoride is a good leaving group; the hydride-transfer mechanism predicts less fluoride ion elimination with the deuterated sample, whereas the proton-transfer mechanism predicts more. Experiments on three types of glyoxalase I (yeast, rat and mouse forms) supported the proton-transfer mechanism in every case. This mechanism was finally observed in crystal structures of glyoxalase I.

Behavior
Multiple studies have reported evidence that Glo1 expression is correlated with differences in anxiety-like behavior in mice as well as behavior in the is_associated_with::tail suspension test, which is sensitive to is_associated_with::antidepressant drugs; however, the direction of these effects have not always been consistent, which has raised skepticism. Differences in Glo1 expression in mice appear to be caused by a is_associated_with::copy number variant that is common among is_associated_with::inbred strains of mice. It has been proposed that the behavioral effects of Glo1 are due to the activity of its principal substrate is_associated_with::methylglyoxal at GABAA receptors. A small molecule inhibitor of glyoxalase I has been shown to have anxiolytic properties, thus identifying another possible indication for inhibitors of Glyoxalase I.

As a drug target
Glyoxalase I is a target for the development of pharmaceuticals against bacteria, protozoans (especially is_associated_with::Trypanosoma cruzi and the is_associated_with::Leishmania) and human cancer. Numerous inhibitors have been developed, most of which share the is_associated_with::glutathione moiety. Among the most tightly binding family of inhibitors to the human enzyme are derivatives of S-(N-aryl-N-hydroxycarbamoyl)glutathione, most notably the p-bromophenyl derivative, which has a is_associated_with::dissociation constant of 14 nM. The closest analog of the transition state is believed to be S-(N-hydroxy-N-p-iodophenylcarbamoyl)glutathione; the crystal structure of this compound bound to the human enzyme has been solved to 2 Å resolution (PDB accession code ).

Experiments suggest that methylglyoxal is preferentially toxic to proliferating cells, such as those in cancer.

Recent research demonstrates that GLO1 expression is upregulated in various human malignant tumors including metastatic melanoma.