Glycolate Oxidase in Complex with the Inhibitor 5-[(4-Chlorophenyl) sulfanyl]-1, 2, 3-thiazadole-4-carboxylate (2W0U) from Homo sapiens
Created by: Morgan Wall
Human glycolate oxidase in complex with the inhibitor 5-[(4-Chlorophenyl) sulfanyl]-1, 2, 3-thiazadole-4-carboxylate (PDB ID: 2W0U) is a flavoprotein found in Homo sapiens. It is one of only two human enzymes in the FMN dependent I-2 hydroxy acid oxidation enzymes (1). Among different species glycolate oxidase differs primarily in the inhibitor it binds and the size of the substrate it is capable of oxidizing. Human glycolate oxidase converts glycolate to glyoxylate and ultimately glyoxylate to oxalate by the process of oxidation (2). The purpose of glycolate oxidation is to break down hydroxy-acids to produce carbohydrate precursors which will ultimately serve as a source of energy. In plants, the oxidation of glycolate reduces the need for photorespiration by providing an alternate source of carbon metabolism (3). While glyoxylate is a necessary precursor for carbohydrates, oxalate accumulation can lead to several diseases including Hyperoxaluria I, making Human glycolate oxidase an important protein for study and a potential therapeutic target. (1).
The primary structure of human glycolate oxidase is composed of 371 amino acid residues organized into 4 polypeptide chains (3). Subunits A, B, and C are homologous monomers while subunit D is unique causing the multimeric unit to be asymmetric. In the functional form of the enzyme, the four polypeptide chains form a tetramer. The protein in complex with the inhibitor has a molecular weight of 163,643 Da and an isoelectric point of 8.44 (4). The isoelectric point is the pH at which the protein has no net electrical charge and is useful for predicting the behavior of the protein in various environments or solvents. Cellular acidity is maintained around a pH of seven while the pH of peroxisomes is significantly lower. The high concentration of hydrogen ions at these pH values allows conserved arginine and lysine residues in the active site to remain protonated. The protonated forms of these residues are important for establishing H bonds with the substrate for stabilization (3).
The secondary structure of human glycolate oxidase in complex with the inhibitor is composed of 42% alpha helices, 13% beta sheets, and random coils. These structures are organized into a tertiary structure consisting primarily of TIM (triosephosphate isomerase) barrels which are stabilized by hydrogen bonds and electrostatic interactions on the side chains of residues in the alpha helices and beta sheets (1). Non-covalent interactions stabilize the structure and create an aliphatic interior with hydrophobic regions between the alpha and beta sheets. The TIM barrel structure is composed of eight alpha helices and eight parallel beta sheets connected by αβ and βα loops. The structure is common among enzymatic proteins of many families and serves as a catalyst for a wide variety of reactions. The active site of the TIM barrel is commonly located at the C terminus of the parallel beta sheets. In human glycolate oxidase this active site will be the site of oxidation for the substrate and ligands (6).
Human glycolate oxidase has two associated ligands. The first ligand is the flavin mononucleotide which is responsible for the oxidation of glycolate to produce glyoxylate. The second ligand 5-[(4-Chlorophenyl) sulfanyl]-1, 2, 3-thiazadole-4-carboxylate functions as an inhibitor of the oxidase by binding to key residues in the active site (8). Five conserved residues contribute to the flavin binding site. This active site is composed of His-260, Lys-236, Thr-158, Gln-130, and Ser-108 (3). The latter four residues all participate in hydrogen bonding with the Flavin mononucleotide in order to stabilize the molecule. Lys-236 forms a hydrogen bond with the N1, O2, and ribityl 2’-OH moieties of the flavin mononucleotide. Thr-158 hydroxyl group on the side chain forms a hydrogen bond with the O2 atom on the flavin mononucleotide. The side chain of Gln-130 hydrogen bonds with O4 on the FMN molecule and the side chain of Ser-108 hydrogen bonds with O4 on the Flavin mononucleotide. Upon binding of the substrate, most commonly glycolate, His-260 functions as a proton acceptor during the oxidation reaction in order to form the carbanion intermediate (3).
Substrate binding sites are located at various residues along the polypeptide chain including Tyr-26, Arg-167, and Arg-263. In the absence of substrate these residues stabilize the inhibitor 5-[(4-Chlorophenyl) sulfanyl]-1, 2, 3-thiazadole-4-carboxylate. When the substrate binds the guanidium group on Arg-167 induces electrostatic interactions that are necessary to stabilize the substrate. Additionally Arg-263 forms hydrogen bonds between the nitrogen atoms in the side chain of the residue and the substrate. Additionally Tyr-26 stabilizes the substrate at the active site through the formation of hydrogen bonds with the side chain hydroxyl group (3). As the substrate binds to these residues via hydrogen bonding, His-260 functions as a proton acceptor and removes the proton from the 2-hydroxy moiety of the substrate (2). As the substrate becomes deprotonated, Lys-236 facilitates a hydride transfer from the alpha carbon to the flavin mononucleotide. Lysine carries a positive charge, which lowers the pKa of a neighboring nitrogen to facilitate the hydride transfer. The transition state of the reaction is stabilized by Tyr-129 and Tyr-132 which bind to the O4 in the flavin mononucleotide (2). Tyrosine is an important residue for this function because of the nature of its side chain. The bulky aromatic ring makes the molecule more stable and the hydroxyl group allows hydrogen bonds to form. This works to stabilize the transition state because a resonance structure forms between the oxygen on the flavin mononucleotide, the hydroxyl oxygen on Tyr-132, and oxygen atoms in the substrate (3).
Normally the flavin mononucleotide site is occluded by the inhibitor. In human glycolate oxidase the inhibitor is the ligand 5-[(4-Chlorophenyl) sulfanyl]-1, 2, 3-thiazadole-4-carboxylate. The inhibitor prevents substrate oxidation by blocking the active site. In order for the inhibitor to be released from the active site, loop 4 must undergo a conformational change causing it to rotate approximately 35 ?. Loop 4 connects the α4 and β4 in the TIM barrel and consists of residues 169-212. Rotation of loop four is induced by the binding of the substrate and removes the inhibitor from the active site allowing the reaction to proceed (2). The mobility of loop four can be noted in the crystallization by using electron density techniques. In the regions of 173-206 the electron density is low compared to other regions of the protein. The decreased electron density indicates that this segment of the protein is highly mobile. It is necessary for the tail of the inhibitor to be mobile in order to increase specificity of substrate binding. Damage to or loss of loop 4 causes a 65% to 70% decrease in specific activity (2).
In order to understand the function of the enzyme and how the structure contributes to the function it is helpful to consider the structures of similar proteins in other organisms. The PSI BLAST program is a useful tool to compare the primary structures of proteins among databases. The PSI BLAST program looks for differences in amino acid sequences and assigns an E value reflecting the level of difference between the primary structures of two proteins. An E value below .5 is considered a significant level of similarity. The Dali Server can be used to compare the tertiary structures of proteins. The Dali Server assigns a Z score based on the number of similarities in the tertiary structures in the two proteins. A Z score above 2 is considered a significant level of similarity. PSI BLAST and the Dali Server both identify the glycolate oxidase found in Spinacia olercea (1AL7) and the hydroxy acid oxidase found in Rattus norvegicus (3SGZ) as proteins with similar structures to human glycolate oxidase. Glycolate oxidase found in Spinacia olercea has an E value of 8 E-166 relative to human glycolate oxidase (5). Additionally the Dali Server assigns glycolate oxidase in Spinacia olercea a Z score of 53 (7). The hydroxy acid oxidase in the species Rattus norvegicus follows a similar pattern with an E value of 2 E-169 and a Z score of 51.5 (5,7).
Human glycolate oxidase, Spinacia olercea glycolate oxidase, and Rattus norvegicus long chain hydroxyacid oxidase all share the common function of substrate oxidation. Due to the nature of this function all three proteins are dependent on the flavin mononucleotide ligand as well as an inhibitory ligand. Spinacia olercea glycolate oxidase is bound to the inhibitor 4-Carboxy-5-(1-Pentyl) Hexylsulfanyl-1,2,3-Triazole and Rattus norvegicus long chain hydroxyacid oxidase binds to the inhibitor5-[(4-methylphenyl) sulfanyl-1, 2, 3-thiadazole-carboxylic acid (3). These proteins all include the same five conserved residues in the active site which allow for the oxidation of the substrate, stability, and the acceptance of the proton.
An important structural and functional difference between the three oxidases are their substrate specificities. As mentioned earlier, the ability of the inhibitor to move plays a key role in determining the specificity of the active site. In human glycolate oxidase, the movement of the inhibitor is dependent on Trp-110. The residue is able to rotate into or away from the active site depending on the substrate bound. This rotation allows for a broad specificity of substrate binding. For smaller substrates the residue functional group rotates inward and rotates outward for larger substrates in order to create space and bind to longer hydroxy acids. The movement of Trp-110 disrupts hydrogen bonds in Leu-191, Tyr-134, and Tyr-208 inducing conformational changes in loop 4 and removing the inhibitor form the active site. Due to the bulky nature of tryptophan, human glycolate oxidase is able to bind to large substrates. Trp-110 is not conserved among species and analogous residues in Spinacia lercea glycolate oxidase and Rattus norvegicus long chain hydroxyacid oxidase do not have the same level of control. Therefore those enzymes are more limited in the range of substrates they are able to bind and tend to bind smaller substrates (2).
The most common substrate bound by human glycolate oxidase is glycolate which become oxidized to produce glyoxylate. Additionally human glycolate oxidase oxidizes glyoxylate to produce oxalate (2). Excess oxalate is normally removed by the enzyme alanine glyoxylate aminotransferase. In the absence of a functional form of this enzyme oxalate accumulates in the liver and binds to calcium. The calcium oxalate compound forms hard crystallized stones, commonly referred to as kidney stones, which can lead to kidney damage or failure (9). Human glycolate has been identified as a therapeutic target for Hyperoxaluria, kidney disease because of its regulatory role in the production of oxalate. Inhibition of the enzymatic activity of Human glycolate oxidase could prevent the production of oxalate and therefore reduce its accumulation in the liver.
Human glycolate oxidase is an important enzyme that should be further investigated as a target for inhibition. The potential to inhibit this flavoprotein would reduce the suffering of those with Hyperoxaluria I and II. The potential to research the enzyme is great due to its presence in a variety of organisms and conserved nature.