Lactoylglutathione Lyase
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The enzyme lactoylglutathione lyase (EC 4.4.1.5, also known as glyoxalase I)
catalyzes Catalysis () is the increase in rate of a chemical reaction due to an added substance known as a catalyst (). Catalysts are not consumed by the reaction and remain unchanged after it. If the reaction is rapid and the catalyst recycles quick ...
the
isomerization In chemistry, isomerization or isomerisation is the process in which a molecule, polyatomic ion or molecular fragment is transformed into an isomer with a different chemical structure. Enolization is an example of isomerization, as is tautomer ...
of hemithioacetal adducts, which are formed in a spontaneous reaction between a glutathionyl group and
aldehyde In organic chemistry, an aldehyde () (lat. ''al''cohol ''dehyd''rogenatum, dehydrogenated alcohol) is an organic compound containing a functional group with the structure . The functional group itself (without the "R" side chain) can be referred ...
s such as
methylglyoxal Methylglyoxal (MGO) is the organic compound with the formula CH3C(O)CHO. It is a reduced derivative of pyruvic acid. It is a reactive compound that is implicated in the biology of diabetes. Methylglyoxal is produced industrially by degradation ...
. :(''R'')-''S''-lactoylglutathione = glutathione + 2-oxopropanal Glyoxalase I derives its name from its catalysis of the first step in the
glyoxalase system The glyoxalase system is a set of enzymes that carry out the detoxification of methylglyoxal and the other reactive aldehydes that are produced as a normal part of metabolism. This system has been studied in both bacteria and eukaryotes. This detox ...
, a critical two-step detoxification system for
methylglyoxal Methylglyoxal (MGO) is the organic compound with the formula CH3C(O)CHO. It is a reduced derivative of pyruvic acid. It is a reactive compound that is implicated in the biology of diabetes. Methylglyoxal is produced industrially by degradation ...
. Methylglyoxal is produced naturally as a byproduct of normal biochemistry, but is highly toxic, due to its chemical reactions with
protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residue (biochemistry), residues. Proteins perform a vast array of functions within organisms, including Enzyme catalysis, catalysing metab ...
s,
nucleic acid Nucleic acids are large biomolecules that are crucial in all cells and viruses. They are composed of nucleotides, which are the monomer components: a pentose, 5-carbon sugar, a phosphate group and a nitrogenous base. The two main classes of nuclei ...
s, and other cellular components. The second detoxification step, in which (''R'')-''S''-lactoylglutathione is split into glutathione and D-lactate, is carried out by
glyoxalase II The enzyme hydroxyacylglutathione hydrolase (EC 3.1.2.6, systematic name = ''S''-(2-hydroxyacyl)glutathione hydrolase) catalysis, catalyzes the following reaction: :''S''-(2-hydroxyacyl)glutathione + H2O = glutathione + a 2-hydroxy carboxylate Th ...
, a
hydrolase In biochemistry, hydrolases constitute a class of enzymes that commonly function as biochemical catalysts that use water to break a chemical bond: :\ce \quad \xrightarrowtext\quad \ce This typically results in dividing a larger molecule into s ...
. Unusually, these reactions carried out by the glyoxalase system does not oxidize glutathione, which usually acts as a
redox Redox ( , , reduction–oxidation or oxidation–reduction) is a type of chemical reaction in which the oxidation states of the reactants change. Oxidation is the loss of electrons or an increase in the oxidation state, while reduction is t ...
coenzyme A cofactor is a non-protein chemical compound or Metal ions in aqueous solution, metallic ion that is required for an enzyme's role as a catalysis, catalyst (a catalyst is a substance that increases the rate of a chemical reaction). Cofactors can ...
. Although
aldose reductase In enzymology, aldose reductase (or aldehyde reductase) () is an enzyme in humans encoded by the gene AKR1B1. It is an cytosolic NADPH-dependent oxidoreductase that catalyzes the reduction of a variety of aldehydes and carbonyls, including monos ...
can also detoxify methylglyoxal, 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
cancer Cancer is a group of diseases involving Cell growth#Disorders, abnormal cell growth with the potential to Invasion (cancer), invade or Metastasis, spread to other parts of the body. These contrast with benign tumors, which do not spread. Po ...
. Several inhibitors of glyoxalase I have been identified, such as S-(N-hydroxy-N-methylcarbamoyl)glutathione. Glyoxalase I is classified as a carbon-sulfur
lyase In biochemistry, a lyase is an enzyme that catalyzes the breaking (an elimination reaction) of various chemical bonds by means other than hydrolysis (a substitution reaction) and oxidation Redox ( , , reduction–oxidation or oxidatio ...
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
redox Redox ( , , reduction–oxidation or oxidation–reduction) is a type of chemical reaction in which the oxidation states of the reactants change. Oxidation is the loss of electrons or an increase in the oxidation state, while reduction is t ...
reaction; one carbon is oxidized whereas the other is reduced. The mechanism proceeds by subtracting and then adding
proton A proton is a stable subatomic particle, symbol , Hydron (chemistry), H+, or 1H+ with a positive electric charge of +1 ''e'' (elementary charge). Its mass is slightly less than the mass of a neutron and approximately times the mass of an e ...
s, forming an enediolate intermediate, rather than by transferring
hydride In chemistry, a hydride is formally the anion of hydrogen (H−), a hydrogen ion with two electrons. In modern usage, this is typically only used for ionic bonds, but it is sometimes (and has been more frequently in the past) applied to all che ...
s. Unusually for a
metalloprotein Metalloprotein is a generic term for a protein that contains a metal ion cofactor. A large proportion of all proteins are part of this category. For instance, at least 1000 human proteins (out of ~20,000) contain zinc-binding protein domains al ...
, this enzyme shows activity with several different metals. Glyoxalase I is also unusual in that it is
stereospecific In chemistry, stereospecificity is the property of a reaction mechanism that leads to different stereoisomeric reaction products from different stereoisomeric reactants, or which operates on only one (or a subset) of the stereoisomers."Overlap C ...
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
yeast Yeasts are eukaryotic, single-celled microorganisms classified as members of the fungus kingdom (biology), kingdom. The first yeast originated hundreds of millions of years ago, and at least 1,500 species are currently recognized. They are est ...
, through
gene duplication Gene duplication (or chromosomal duplication or gene amplification) is a major mechanism through which new genetic material is generated during molecular evolution. It can be defined as any duplication of a region of DNA that contains a gene ...
.


Nomenclature

The
systematic name A systematic name is a name given in a systematic way to one unique group, organism, object or chemical substance, out of a specific population or collection. Systematic names are usually part of a nomenclature. A semisystematic name or semitrivi ...
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
trypanothione Trypanothione is an unusual form of glutathione containing two molecules of glutathione joined by a spermidine (polyamine) linker. It is found in parasitic protozoa such as leishmania and trypanosomes. These protozoal parasites are the cause of le ...
, the analog of glutathione in parasitic protozoa such as the trypanosomes. The human gene for this enzyme is called
GLO1 The enzyme lactoylglutathione lyase (EC 4.4.1.5, also known as glyoxalase I) catalyzes the isomerization of hemithioacetal adducts, which are formed in a spontaneous reaction between a glutathionyl group and aldehydes such as methylglyoxal. :( ...
.


Gene

Lactoylglutathione lyase in humans is encoded by the ''GLO1''
gene In biology, the word gene has two meanings. The Mendelian gene is a basic unit of heredity. The molecular gene is a sequence of nucleotides in DNA that is transcribed to produce a functional RNA. There are two types of molecular genes: protei ...
.


Structure

Several
structures A structure is an arrangement and organization of interrelated elements in a material object or system, or the object or system so organized. Material structures include man-made objects such as buildings and machines and natural objects such as ...
of glyoxalase I have been solved. Four structures of the human form have been published, with PDB accession codes , , , and . Five structures of the ''
Escherichia coli ''Escherichia coli'' ( )Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. is a gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus '' Escherichia'' that is commonly fo ...
'' form have been published, with accession codes , , , , and . Finally, one structure of the trypanothione-specific version from ''
Leishmania major ''Leishmania major'' is a species of parasite found in the genus ''Leishmania'', and is associated with the disease zoonotic cutaneous leishmaniasis (also known as Aleppo boil, Baghdad boil, Bay sore, Biskra button, Chiclero ulcer, Delhi boil, Ka ...
'' has been solved, . In all these cases, the
quaternary structure Protein quaternary structure is the fourth (and highest) classification level of protein structure. Protein quaternary structure refers to the structure of proteins which are themselves composed of two or more smaller protein chains (also refe ...
of the biological unit is a domain-swapped dimer, in which the active site and the 8-stranded
beta sheet The beta sheet (β-sheet, also β-pleated sheet) is a common motif of the regular protein secondary structure. Beta sheets consist of beta strands (β-strands) connected laterally by at least two or three backbone hydrogen bonds, forming a gene ...
secondary structure Protein secondary structure is the local spatial conformation of the polypeptide backbone excluding the side chains. The two most common Protein structure#Secondary structure, secondary structural elements are alpha helix, alpha helices and beta ...
is formed from both subunits. However, in
yeast Yeasts are eukaryotic, single-celled microorganisms classified as members of the fungus kingdom (biology), kingdom. The first yeast originated hundreds of millions of years ago, and at least 1,500 species are currently recognized. They are est ...
such as ''
Saccharomyces cerevisiae ''Saccharomyces cerevisiae'' () (brewer's yeast or baker's yeast) is a species of yeast (single-celled fungal microorganisms). The species has been instrumental in winemaking, baking, and brewing since ancient times. It is believed to have be ...
'', the two subunits have fused into a single monomer of double size, through
gene duplication Gene duplication (or chromosomal duplication or gene amplification) is a major mechanism through which new genetic material is generated during molecular evolution. It can be defined as any duplication of a region of DNA that contains a gene ...
. Each half of the structural dimer is a sandwich of 3-4
alpha helices An alpha helix (or α-helix) is a sequence of amino acids in a protein that are twisted into a coil (a helix). The alpha helix is the most common structural arrangement in the secondary structure of proteins. It is also the most extreme type of l ...
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
fosfomycin Fosfomycin, sold under the brand name Monurol among others, is an antibiotic primarily used to treat lower urinary tract infections. It is not indicated for kidney infections. Occasionally it is used for prostate infections. It is generally ...
,
bleomycin -13- (1''H''-imidazol-5-yl)methyl9-hydroxy-5- 1''R'')-1-hydroxyethyl8,10-dimethyl-4,7,12,15-tetraoxo-3,6,11,14-tetraazapentadec-1-yl}-2,4'-bi-1,3-thiazol-4-yl)carbonyl]amino}propyl)(dimethyl)sulfonium , C=55 , H=84 , N=17 , O=21 , S=3 , SMI ...
and
mitomycin The mitomycins are a family of aziridine-containing Secondary metabolite, natural products isolated from ''Streptomyces caespitosus'' or ''Streptomyces lavendulae.'' They include mitomycin A, mitomycin B, and mitomycin C. When the name mitomycin ...
. Likewise, the unrelated enzymes
methylmalonyl-CoA epimerase Methylmalonyl-CoA is the thioester consisting of coenzyme A linked to methylmalonic acid. It is an important intermediate in the biosynthesis of succinyl-CoA, which plays an essential role in the tricarboxylic acid cycle (aka the Citric Acid Cycle ...
,
3-demethylubiquinone-9 3-O-methyltransferase In enzymology, a 3-demethylubiquinone-9 3-O-methyltransferase () is an enzyme that catalyzes the chemical reaction :S-adenosyl-L-methionine + 3-demethylubiquinone-9 \rightleftharpoons S-adenosyl-L-homocysteine + ubiquinone-9 Thus, the two subst ...
and numerous
dioxygenase Dioxygenases are oxidoreductase enzymes. Aerobic organism, Aerobic life, from simple single-celled bacteria species to complex eukaryotic organisms, has evolved to depend on the oxidizing power of dioxygen in various metabolic pathways. From energ ...
s such as biphenyl-2,3-diol 1,2-dioxygenase, catechol 2,3-dioxygenase, 3,4-dihydroxyphenylacetate 2,3-dioxygenase and
4-hydroxyphenylpyruvate dioxygenase 4-Hydroxyphenylpyruvate dioxygenase (HPPD), also known as α-ketoisocaproate dioxygenase (KIC dioxygenase), is an Fe(II)-containing non-heme oxygenase that catalyzes the second reaction in the catabolism of tyrosine - the conversion of 4- ...
all resemble glyoxalase I in structure. Finally, many proteins of unknown or uncertain function likewise resemble glyoxalase I, such as At5g48480 from the plant, ''
Arabidopsis thaliana ''Arabidopsis thaliana'', the thale cress, mouse-ear cress or arabidopsis, is a small plant from the mustard family (Brassicaceae), native to Eurasia and Africa. Commonly found along the shoulders of roads and in disturbed land, it is generally ...
''. The active site has four major regions.


Function

The principal physiological function of glyoxalase I is the detoxification of
methylglyoxal Methylglyoxal (MGO) is the organic compound with the formula CH3C(O)CHO. It is a reduced derivative of pyruvic acid. It is a reactive compound that is implicated in the biology of diabetes. Methylglyoxal is produced industrially by degradation ...
, a reactive
2-oxoaldehyde In organic chemistry, a dicarbonyl is a molecule containing two carbonyl () groups. Although this term could refer to any organic compound containing two carbonyl groups, it is used more specifically to describe molecules in which both carbonyls ...
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
threonine Threonine (symbol Thr or T) is an amino acid that is used in the biosynthesis of proteins. It contains an α-amino group (which is in the protonated −NH form when dissolved in water), a carboxyl group (which is in the deprotonated −COO− ...
. To minimize the amount of toxic methylglyoxal and other reactive 2-oxoaldehydes, the
glyoxalase system The glyoxalase system is a set of enzymes that carry out the detoxification of methylglyoxal and the other reactive aldehydes that are produced as a normal part of metabolism. This system has been studied in both bacteria and eukaryotes. This detox ...
has evolved. The methylglyoxal reacts spontaneously with reduced
glutathione Glutathione (GSH, ) is an organic compound with the chemical formula . It is an antioxidant in plants, animals, fungi, and some bacteria and archaea. Glutathione is capable of preventing damage to important cellular components caused by sources ...
(or its equivalent,
trypanothione Trypanothione is an unusual form of glutathione containing two molecules of glutathione joined by a spermidine (polyamine) linker. It is found in parasitic protozoa such as leishmania and trypanosomes. These protozoal parasites are the cause of le ...
),) 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
glyoxalase II The enzyme hydroxyacylglutathione hydrolase (EC 3.1.2.6, systematic name = ''S''-(2-hydroxyacyl)glutathione hydrolase) catalysis, catalyzes the following reaction: :''S''-(2-hydroxyacyl)glutathione + H2O = glutathione + a 2-hydroxy carboxylate Th ...
. The former enzyme, described here, rearranges the hemithioacetal formed naturally by the attack of
glutathione Glutathione (GSH, ) is an organic compound with the chemical formula . It is an antioxidant in plants, animals, fungi, and some bacteria and archaea. Glutathione is capable of preventing damage to important cellular components caused by sources ...
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
coenzyme A cofactor is a non-protein chemical compound or Metal ions in aqueous solution, metallic ion that is required for an enzyme's role as a catalysis, catalyst (a catalyst is a substance that increases the rate of a chemical reaction). Cofactors can ...
and is required only in catalytic (i.e., very small) amounts; normally, glutathione acts instead as a
redox Redox ( , , reduction–oxidation or oxidation–reduction) is a type of chemical reaction in which the oxidation states of the reactants change. Oxidation is the loss of electrons or an increase in the oxidation state, while reduction is t ...
couple in oxidation-reduction reactions. The glyoxalase system has also been suggested to play a role in regulating cell growth and in assembling
microtubule Microtubules are polymers of tubulin that form part of the cytoskeleton and provide structure and shape to eukaryotic cells. Microtubules can be as long as 50 micrometres, as wide as 23 to 27 nanometer, nm and have an inner diameter bet ...
s.


Properties

Glyoxalase I requires bound metal ions for catalysis. The human enzyme and its counterparts in yeast (''
Saccharomyces cerevisiae ''Saccharomyces cerevisiae'' () (brewer's yeast or baker's yeast) is a species of yeast (single-celled fungal microorganisms). The species has been instrumental in winemaking, baking, and brewing since ancient times. It is believed to have be ...
'') and ''
Pseudomonas putida ''Pseudomonas putida'' is a Gram-negative, rod-shaped, saprophytic soil bacterium. It has a versatile metabolism and is amenable to genetic manipulation, making it a common organism used in research, bioremediation, and synthesis of chemicals and ...
'' use divalent
zinc Zinc is a chemical element; it has symbol Zn and atomic number 30. It is a slightly brittle metal at room temperature and has a shiny-greyish appearance when oxidation is removed. It is the first element in group 12 (IIB) of the periodic tabl ...
, Zn2+. By contrast, the prokaryotic versions often use a
nickel Nickel is a chemical element; it has symbol Ni and atomic number 28. It is a silvery-white lustrous metal with a slight golden tinge. Nickel is a hard and ductile transition metal. Pure nickel is chemically reactive, but large pieces are slo ...
ion. The glyoxalase I found in eukaryotic trypanosomal parasites such as ''
Leishmania major ''Leishmania major'' is a species of parasite found in the genus ''Leishmania'', and is associated with the disease zoonotic cutaneous leishmaniasis (also known as Aleppo boil, Baghdad boil, Bay sore, Biskra button, Chiclero ulcer, Delhi boil, Ka ...
'' and ''
Trypanosoma cruzi ''Trypanosoma cruzi'' is a species of parasitic euglenoids. Among the protozoa, the trypanosomes characteristically bore tissue in another organism and feed on blood (primarily) and also lymph. This behaviour causes disease or the likelihood ...
'' can also use nickel for activity, possibly reflecting an acquisition of their GLO1 gene by
horizontal gene transfer Horizontal gene transfer (HGT) or lateral gene transfer (LGT) is the movement of genetic material between organisms other than by the ("vertical") transmission of DNA from parent to offspring (reproduction). HGT is an important factor in the e ...
. A property of glyoxalase I is its lack of specificity for the catalytic metal ion. Most enzymes bind one particular type of metal, and their catalytic activity depends on having bound that metal. For example,
oxidoreductase In biochemistry, an oxidoreductase is an enzyme that catalyzes the transfer of electrons from one molecule, the reductant, also called the electron donor, to another, the oxidant, also called the electron acceptor. This group of enzymes usually ut ...
s often use a specific
metal A metal () is a material that, when polished or fractured, shows a lustrous appearance, and conducts electrical resistivity and conductivity, electricity and thermal conductivity, heat relatively well. These properties are all associated wit ...
ion such as
iron Iron is a chemical element; it has symbol Fe () and atomic number 26. It is a metal that belongs to the first transition series and group 8 of the periodic table. It is, by mass, the most common element on Earth, forming much of Earth's o ...
,
manganese Manganese is a chemical element; it has Symbol (chemistry), symbol Mn and atomic number 25. It is a hard, brittle, silvery metal, often found in minerals in combination with iron. Manganese was first isolated in the 1770s. It is a transition m ...
or
copper Copper is a chemical element; it has symbol Cu (from Latin ) and atomic number 29. It is a soft, malleable, and ductile metal with very high thermal and electrical conductivity. A freshly exposed surface of pure copper has a pinkish-orang ...
and will fail to function if their preferred metal ion is replaced, due to differences in the
redox potential Redox potential (also known as oxidation / reduction potential, ''ORP'', ''pe'', ''E_'', or E_) is a measure of the tendency of a chemical species to acquire electrons from or lose electrons to an electrode and thereby be reduced or oxidised respe ...
; thus, the ferrous
superoxide dismutase Superoxide dismutase (SOD, ) is an enzyme that alternately catalyzes the dismutation (or partitioning) of the superoxide () anion radical into normal molecular oxygen (O2) and hydrogen peroxide (). Superoxide is produced as a by-product of oxy ...
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
magnesium Magnesium is a chemical element; it has Symbol (chemistry), symbol Mg and atomic number 12. It is a shiny gray metal having a low density, low melting point and high chemical reactivity. Like the other alkaline earth metals (group 2 ...
,
manganese Manganese is a chemical element; it has Symbol (chemistry), symbol Mn and atomic number 25. It is a hard, brittle, silvery metal, often found in minerals in combination with iron. Manganese was first isolated in the 1770s. It is a transition m ...
,
cobalt Cobalt is a chemical element; it has Symbol (chemistry), symbol Co and atomic number 27. As with nickel, cobalt is found in the Earth's crust only in a chemically combined form, save for small deposits found in alloys of natural meteoric iron. ...
,
nickel Nickel is a chemical element; it has symbol Ni and atomic number 28. It is a silvery-white lustrous metal with a slight golden tinge. Nickel is a hard and ductile transition metal. Pure nickel is chemically reactive, but large pieces are slo ...
and even
calcium Calcium is a chemical element; it has symbol Ca and atomic number 20. As an alkaline earth metal, calcium is a reactive metal that forms a dark oxide-nitride layer when exposed to air. Its physical and chemical properties are most similar to it ...
.; 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
cadmium Cadmium is a chemical element; it has chemical symbol, symbol Cd and atomic number 48. This soft, silvery-white metal is chemically similar to the two other stable metals in group 12 element, group 12, zinc and mercury (element), mercury. Like z ...
; however, the enzyme is inert with bound zinc, due to a change in
coordination geometry The coordination geometry of an atom is the geometrical pattern defined by the atoms around the central atom. The term is commonly applied in the field of inorganic chemistry, where diverse structures are observed. The coordination geometry depen ...
from
octahedral In geometry, an octahedron (: octahedra or octahedrons) is any polyhedron with eight faces. One special case is the regular octahedron, a Platonic solid composed of eight equilateral triangles, four of which meet at each vertex. Many types of i ...
to
trigonal bipyramidal In chemistry, a trigonal bipyramid formation is a molecular geometry with one atom at the center and 5 more atoms at the corners of a triangular bipyramid. This is one geometry for which the bond angles surrounding the central atom are not identi ...
. 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
glutamate Glutamic acid (symbol Glu or E; known as glutamate in its anionic form) is an α-amino acid that is used by almost all living beings in the biosynthesis of proteins. It is a Essential amino acid, non-essential nutrient for humans, meaning that ...
s 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.


Reaction mechanism

The
methylglyoxal Methylglyoxal (MGO) is the organic compound with the formula CH3C(O)CHO. It is a reduced derivative of pyruvic acid. It is a reactive compound that is implicated in the biology of diabetes. Methylglyoxal is produced industrially by degradation ...
molecule consists of two
carbonyl In organic chemistry, a carbonyl group is a functional group with the formula , composed of a carbon atom double bond, double-bonded to an oxygen atom, and it is divalent at the C atom. It is common to several classes of organic compounds (such a ...
groups flanked by a
hydrogen Hydrogen is a chemical element; it has chemical symbol, symbol H and atomic number 1. It is the lightest and abundance of the chemical elements, most abundant chemical element in the universe, constituting about 75% of all baryon, normal matter ...
atom and a
methyl In organic chemistry, a methyl group is an alkyl derived from methane, containing one carbon atom bonded to three hydrogen atoms, having chemical formula (whereas normal methane has the formula ). In formulas, the group is often abbreviated as ...
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
glutathione Glutathione (GSH, ) is an organic compound with the chemical formula . It is an antioxidant in plants, animals, fungi, and some bacteria and archaea. Glutathione is capable of preventing damage to important cellular components caused by sources ...
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
glutathione Glutathione (GSH, ) is an organic compound with the chemical formula . It is an antioxidant in plants, animals, fungi, and some bacteria and archaea. Glutathione is capable of preventing damage to important cellular components caused by sources ...
— probably in its reactive
thiolate In organic chemistry, a thiol (; ), or thiol derivative, is any organosulfur compound of the form , where R represents an alkyl or other organic substituent. The functional group itself is referred to as either a thiol group or a sulfhydryl grou ...
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
glutamate Glutamic acid (symbol Glu or E; known as glutamate in its anionic form) is an α-amino acid that is used by almost all living beings in the biosynthesis of proteins. It is a Essential amino acid, non-essential nutrient for humans, meaning that ...
s, a
histidine Histidine (symbol His or H) is an essential amino acid that is used in the biosynthesis of proteins. It contains an Amine, α-amino group (which is in the protonated –NH3+ form under Physiological condition, biological conditions), a carboxylic ...
and one other sidechain, usually another histidine or
glutamate Glutamic acid (symbol Glu or E; known as glutamate in its anionic form) is an α-amino acid that is used by almost all living beings in the biosynthesis of proteins. It is a Essential amino acid, non-essential nutrient for humans, meaning that ...
s. 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. It is worthy to note that the first theoretically confirmed mechanism for the ''R''-substrate of glyoxalase one published recently. The catalytic mechanism of Glyoxalase has been studied by density functional theory, molecular dynamics simulations and hybrid QM/MM methods. The reason for the special specificity of the enzyme (it accepts both enantiomers of its chiral substrate but converts them to the same enantiomer of the product) is the higher basicity and flexibility of one of the active site glutamates (Glu172).


Proton vs. hydride transfer

Glyoxalase I was originally believed to operate by the transfer of a
hydride In chemistry, a hydride is formally the anion of hydrogen (H−), a hydrogen ion with two electrons. In modern usage, this is typically only used for ionic bonds, but it is sometimes (and has been more frequently in the past) applied to all che ...
, which is a
proton A proton is a stable subatomic particle, symbol , Hydron (chemistry), H+, or 1H+ with a positive electric charge of +1 ''e'' (elementary charge). Its mass is slightly less than the mass of a neutron and approximately times the mass of an e ...
surrounded by two
electron The electron (, or in nuclear reactions) is a subatomic particle with a negative one elementary charge, elementary electric charge. It is a fundamental particle that comprises the ordinary matter that makes up the universe, along with up qua ...
s (H). In this, it was thought to resemble the classic
Cannizzaro reaction The Cannizzaro reaction, named after its discoverer Stanislao Cannizzaro, is a chemical reaction which involves the base-induced disproportionation of two molecules of a non-enolizable aldehyde to give a primary alcohol and a carboxylic acid. ...
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
proton A proton is a stable subatomic particle, symbol , Hydron (chemistry), H+, or 1H+ with a positive electric charge of +1 ''e'' (elementary charge). Its mass is slightly less than the mass of a neutron and approximately times the mass of an e ...
(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, 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 isotope effect on substrates
fluorinated In chemistry, halogenation is a chemical reaction which introduces one or more halogens into a chemical compound. Halide-containing compounds are pervasive, making this type of transformation important, e.g. in the production of polymers, drugs. ...
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.


Clinical significance


Behavior

''Glo1'' expression is correlated with differences in anxiety-like behavior in mice as well as behavior in the
tail suspension test The tail suspension test (TST) is an experimental method used in scientific research to measure stress in rodents. It is based on the observation that if a mouse is subjected to short term inescapable stress then the mouse will become immobi ...
, which is sensitive to
antidepressant Antidepressants are a class of medications used to treat major depressive disorder, anxiety disorders, chronic pain, and addiction. Common side effects of antidepressants include Xerostomia, dry mouth, weight gain, dizziness, headaches, akathi ...
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 copy number variant that is common among
inbred strain Inbred strains (also called inbred lines, or rarely for animals linear animals) are individuals of a particular species which are nearly identical to each other in genotype due to long inbreeding. A strain is generally defined to be inbred once it ...
s of mice. It has been proposed that the behavioral effects of ''Glo1'' are due to the activity of its principal substrate
methylglyoxal Methylglyoxal (MGO) is the organic compound with the formula CH3C(O)CHO. It is a reduced derivative of pyruvic acid. It is a reactive compound that is implicated in the biology of diabetes. Methylglyoxal is produced industrially by degradation ...
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 ''
Trypanosoma cruzi ''Trypanosoma cruzi'' is a species of parasitic euglenoids. Among the protozoa, the trypanosomes characteristically bore tissue in another organism and feed on blood (primarily) and also lymph. This behaviour causes disease or the likelihood ...
'' and the ''
Leishmania ''Leishmania'' () is a genus of parasitic protozoans, single-celled eukaryotic organisms of the trypanosomatid group that are responsible for the disease leishmaniasis. The parasites are transmitted by sandflies of the genus '' Phlebotomus'' ...
'') and human cancer. Numerous inhibitors have been developed, most of which share the
glutathione Glutathione (GSH, ) is an organic compound with the chemical formula . It is an antioxidant in plants, animals, fungi, and some bacteria and archaea. Glutathione is capable of preventing damage to important cellular components caused by sources ...
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
dissociation constant In chemistry, biochemistry, and pharmacology, a dissociation constant (''K''D) is a specific type of equilibrium constant that measures the propensity of a larger object to separate (dissociate) reversibly into smaller components, as when a complex ...
of 14 nM. The closest analog of the
transition state In chemistry, the transition state of a chemical reaction is a particular configuration along the reaction coordinate. It is defined as the state corresponding to the highest potential energy along this reaction coordinate. It is often marked w ...
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.


References


Further reading

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External links

* * {{Portal bar, Biology, border=no EC 4.4.1 Zinc enzymes Nickel enzymes Enzymes of known structure