Lactate Dehydrogenase
Created by Katelyn Nguyen
Lactate dehydrogenase(LDH) is an enzyme that catalyzes the interconversion of pyruvate and lactate and the associated conversion of NADH to NAD+ (refer to Image 1.) (12) It has a molecular weight of 35735.44 g/mol, an isoelectric point of 7.12 and a 322 residue amino acid sequence within one chain ( Chain A) (2.) This particular enzyme contains two specific ligands: glycerol and NADH with the latter having a greater effect on the functionality of the molecule by serving as the primary cofactor. Glycerol has no known functionality in this particular enzyme except for crystallization purposes. The lactate dehydrogenase of Plasmodium falciparum (pdb id= 1T2C), a type of parasite and agent of malaria shares similar molecular and structural functions with human lactate dehydrogenase despite the differences in amino acid sequences. A further look at its secondary structure indicates that it's 47% helical (17 helices and 154 residues), 19% beta sheets (15 strands and 63 residues) and the remaining 34%(105 residues) are random coils in the sequence (9.) The domain of the pfLDH enzyme consists of 149 residues that are parallel beta sheets and 166 residues of antiparallel beta sheets (segregated alpha and beta regions.) Due to the functional similarities, P. falciparum lactate dehydrogenase is often used in place of human LDH to determine the functional effects of various therapeutic treatments (12.) One type of treatment allows competitive inhibitors to interact with the amino acids of the active site of the parasite LDH and nicotinamide ring of the NADH cofactor leading to sequence changes and providing way for new anti-malarial drug treatments.
Plasmodium parasites lack functional Krebs cycles so they rely extensively on the anaerobic fermentation of glucose as their source of energy via ATP production. Plasmodium falciparum depends on glucose metabolism for its ATP supply so the enzymes of glycolysis are often expressed at high levels and represent potential drug targets. Lactate dehydrogenase is one example of an enzyme that's crucial to the energy production and survival of P. falciparum. It serves as a 2-hydroxy acid oxidoreductase (redox reaction) that converts pyruvate to L-lactate while regenerating NAD+ for continued use in glycolysis (refer to Image 1) (2.) LDH ensures the regeneration of NAD+ from NADH to carry out further glycolysis and energy production for the cells. As a result, the inhibition of LDH will stop the production of ATP and lead to the eventual cell death of the Plasmodium parasites.
The active site of pfLDH has unique properties due to its amino acid sequence and is also the target of many inhibitors (11.) PfLDH (pdb id=1t2c) still retains the catalytic residues D-168, H-195, R-171, and R-109 that are also found in mammalian LDH, but conserved residues that define the active site such as Q102 is replaced by a lysine residue in pfLDH. Similarly, T-246 and I-250 that define the cofactor binding site in most LDH are replaced by proline residues in pfLDH (1.) PfLDH also has a unique five-amino-acid extension consisting of aspartic acid,lysine, glutamic acid, tryptophan, and asparagine in the loop of amino acids that defines the substrate site (1.) This five residue insertion in the active site loop closes down on the active site during catalysis and the displacement (~1.2Ao) of the nicotinamide ring of the NADH cofactor (3.) These changes in the amino acid sequence help define the lactate dehydrogenase enzyme of P. falciparum and provide the basis for a unique set of properties that distinguish it from other LDH isoforms.
Furthermore,the NADH ligand acts as a cofactor for the enzyme whose displacement providesthe enzyme its unique biochemical properties (11.) The NADH cofactor is buried deep within the ternary complex in an active, but closed conformation for greater substrate specificity (11.) The nicotinamide end of the cofactor closest to the substrate and catalytic center constantly changes the position of the NADH relative to the protein resulting in a change to the kinetics of catalysis and substrate inhibition. The position of the atoms of the nicotinamide ring near the substrate and catalytic center differ by 1.2 Ao prompting changes in the NADH cofactor (1.) When NADH and pyruvate bind to the active site of the PfLDH, they form a ternary complex followed by a rate-determining conformational change that causes the substrate specificity loop to close around the complex (refer to Image 2.) This conformational change brings R109 (a catalytic residue) into the active site to polarize the ketone carbonyl of the pyruvate. A hydride is transferred fromNADH to the polarized ketone group with the help of the proton donation from the D-168/H-195 residues. R-171 interacts with the carboxylate functional group to help anchor the pyruvate to the enzyme. Together, residues 98-109 form a loop around the ternary complex creating the catalytic site where pyruvate is converted to lactate (1.) Serine-245 found in malarial LDH is crucial for the correct functioning of the pfLDH enzyme because of its role in the creation of the pyruvate-binding site and the binding of the NADH in the active site (2.) Thus, mutation of the Ser-245 residue reduces the catalytic activity of the enzyme (2.) The rearrangement of the NADH cofactor in association with the protein will change its kinetics of catalysis and substrate inhibition prompting the differences between mammalian and malarial lactate dehydrogenase.
The lactate dehydrogenase of Plasmodium falciparum (pdb id = 1T2C) shares a 29% sequence identity with the human LDH-B A chain (pdb id = 1T2F) with both types conserving the same catalytic residues (Arg-109, Arg-171, His-195 and Asp-168), despite the fact that the human isoform is a much bigger molecule with 4 separate chains. However, there are five positions that differentiate the mammalian and malarial enzymes which include the 245-247 loop and positions 163 and 107 (part of the active site loop).These residues cluster around the nicotinamide binding site of the cofactor pocket, an area that many selective inhibitors tend to bind (3.) A comparison of the structures of P. falciparum and human LDH shows two differences: positioning of the NADH cofactor leads to a sequence change in the cofactor binding pocket that displaces the nicotinamide ring by about 1.2 A as well as a change in the loop region that closes down on the active site during catalysis (2.) Another major difference between the human and parasitic isoforms of LDH involves substrate inhibition and the formation of a covalent complex between pyruvate and NAD+ at the active site (1.) Substrate inhibition is caused by the slow release of the reduced cofactor NAD+ and the formation of a covalent adduct with pyruvate within the active site due to the single point mutation of serine 163 to leucine (3.) For human LDH and other non-parasitic LDH, low concentrations of pyruvate causes substrate inhibition due to the slow cofactor release and the formation of a NAD+-pyruvate complex during the catalytic cycle. In human LDH, the hydroxyl group of S163 forms a bridging water molecule with the amide of the nicotinamide moiety in NADH. The serine hydroxyl forms a hydrogen bond with the amine group of the nicotinamide cofactor and a water bridge with the adjacent G164 (3.) This arrangement reduces the rate of release of NAD+ after the reduction of pyruvate and release of lactate thus forming an NAD+- pyruvate complex (lower the kinetics of the reaction) (1.) In pfLDH (pdb id: 1T2C), however, a mutation causes leucine residues to replace all of the serine residues and remove the substrate inhibition (1.) The bulky, hydrophobic leucine side chain reduces the interaction and disrupts the conserved water binding site. As a result, the nicotinamide ring is placed closer to the main chain and into the pyruvate binding site (altered placement of the nicotinamide ring creates a less favorable environment for adduct stabilization) (1.) Thus, the loss of substrate inhibition is due to the weaker binding of pyruvate to the enzyme-NAD+complex allowing for increased kinetics of the reaction (1.) As a result, pfLDH and other forms of pLDH lack substrate inhibition even at high levels of pyruvate, an adaptation that allows these organisms to survive in pyruvate-rich conditions in the red blood cell (11.)
The catalytic cavity of pfLDH near the nicotinamide moiety has three main features that promote the changes in cofactor association. First, in most LDH, Serine-163 protrudes into the nicotinamide binding site where the nitrogen of the nicotinamide side chain binds to and forms a water bridge with adjacent Gly-164. In pfLDH, Ser-163 is replaced by Leu-163 where an oxygen atom acts as proton acceptor and hydrogen bonds with the nitrogen of thenicotinamide side chain. Second, the planar nicotinamide ring tends to arrange itself in the hydrophobic surface near the side chain of Ile-250 of the regularLDH structures. However, this residue is replaced by a proline whose trans conformation causes it to extend itself into the catalytic cavity and allow the appropriate placement of the cofactor without steric hindrance (11.) Third, residue T-246 near the nicotinamide moiety and substrate site is substituted with a proline in pfLDH causing rearrangement of the neighboring residue, S-245 that's directly bonded to the substrate analogue (11.)
The amino acid sequence differences that distinguish pfLDH from its mammalian form also cause it to become targets of selective inhibitors many of which show signs of antimalarial activities. Inhibitors can bind to the active site of pfLDH in one of three ways: to the pyruvate site, the bridging site of the cofactor and pyruvate, or the adenine binding site (4.) Inhibitors tend to bind to the dinucleotide fold (Rossmann fold) of pLDH at thecofactor binding site in a perpendicular orientation of the plane (1.) Inhibitors bind to the nicotinamide residue binding region of the active site and shares interactions with the substrate-binding residue. Thus, by utilizing the active-site interactions with the cofactor and substrate-binding residues, the inhibitors can enhance their binding affinity and selectivity for the particular enzyme which in this case is lactate dehydrogenase (1.)
Natural product gossypol is an inhibitor of pfLDH by binding to the nicotinamide-ribose site (cofactor binding site) of the protein adjacent to the cleft and is often used in anti-malarial treatments (11.) Gossypol and its derivatives mimic the pyruvate/nicotinamide ribose moiety of NADH and easily bind to the pfLDH protein to minimize the energy of the structure (refer to Image 3) (10.) Gossypol inhibits LDH at sub-molecular levels by competing for NADH which is detrimental to the survival of the Plasmodium because the regeneration of NAD+from NADH is needed to allow glycolysis to continue and produce additional pyruvate for energy (2.) Gossypol derivatives tend to bind to the bridging site for the nicotinamide of the cofactor and the pyruvate located in the active site of the pfLDH. Glycolytic enzymes of pfLDH are oftentimes potential targets for the development of new drugs especially those that have unique features compared to their human counterparts such as the structural design of the active-sites. A series of heterocyclic, azole-based compounds have also been shown to selectively bind with the active site to inhibit the function of the lactate dehydrogenase (refer to Image 4) (5.) More specifically, the azole inhibitors bind directly with the active site of lactate dehydrogenase enzyme where the keto acid (pyruvate) substrate usually attaches to. When an azole inhibitor is bound within the active site, the loop adopts a “closed” conformation with Ser 245 pointing directly into the active site and forming a hydrogen bond with the inhibitor (3.) Inhibitors form an interaction with amino acids surrounding the nicotinamide ring of the NAD+ cofactor of parasite strains such as Plasmodium and prevent the catalysis of homolactic fermentation of pyruvate to lactate (2.) By targeting enzymes that are used in glycolysis, ATP production would be reduced which would lead to the death of the parasites.
Furthermore,the pLDH from three other species of human malarial parasites (Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae) share 90-92% structural and kinetic properties of P. falciparum. Highly conserved residues such as G-27,G-29, G-32, D-65, A-98, N-116, N-140, P-141, D-143, S-153, G-154, G-162, L-167, and E-311 are found in all four species of pLDH (1.) Furthermore, they all have identical catalytic residues which are aspartic acid (D-168), histidine (H-195), and arginine (R-109, R-171.) In contrast, there are 17 highly conserved LDH residues that are not preserved in pLDH. However, not all changes to these active-site residues in one species cause identical changes in the other pLDH. There are 50 sites in the amino sequence that have residue differences among the four pLDH. For example, conserved residue K-243 is a histidine in pfLDH, poLDH and pmLDD, but a leucine in pvLDH (pdb id: 2A92) (6.) However, the one significant feature of all four species of pLDH is that they all have the same five-amino acid insert (DKEWN) in the substrate specificity loops immediately in front of the conserved catalytic residue R-109 (1.) Thus, inhibitors binding to the cofactor sites of these pLDH have similar orientations in all four species where the docked ligands are positioned at the nicotinamide end of the cofactor site (1.) The development of anti-malarials tries to target all four species of parasites especially with the emerging drug resistance patterns that one may have over the other. A comparison of the cofactor properties of the four pLDH with NADH, NAD+, and 3-acetylpyridine adenine dinucleotide (analogue of NAD+) indicates that all four pLDH use APAD+ more efficiently than NAD+due to the high kcat values and greater catalytic efficiencies (refer to Image 4) (1.) APAD+ bound to the enzyme causes faster conformational change and movement of the substrate specificity loop (rate-determining step) in the catalytic cycle of LDH than with NAD+ to allow the pyruvate to adhere to the enzyme (1.) As a result, APAD+ binds more readily in the absence of the Ser-163 residue. Additionally, APAD+ has a higher oxidation potential than NAD+ so the hydride transfer would be faster with APAD+than with NAD+ (increased kinetics between the two cofactors) (1) and faster release of pyruvate (4.) Human LDH, on the other hand, functions better with NAD+ than with the APAD+ analogue as the cofactor unit (1.) At high levels of lactate, pLDHs are five-hundred times more active with APAD than human LDH (3.)
Lactate dehydrogenase contains many different binding sites that react differently to different substrates, but is essentially useful in glycolysis pathways and the production of energy for the cells. Its unique active sites and cofactor features make it a useful target of anti-malarial treatments with the use of inhibitors. Further research is still being conducted on this particular enzyme, but current studies have already shown its tremendous therapeutic potential in the fight against malaria.