A highly prevalent equine glycogen storage disease is explained by constitutive activation of a mutant glycogen synthase.

BACKGROUND: Equine type 1 polysaccharide storage myopathy (PSSM1) is associated with a missense mutation (R309H) in the glycogen synthase (GYS1) gene, enhanced glycogen synthase (GS) activity and excessive glycogen and amylopectate inclusions in muscle. METHODS: Equine muscle biochemical and recombinant enzyme kinetic assays in vitro and homology modelling in silico, were used to investigate the hypothesis that higher GS activity in affected horse muscle is caused by higher GS expression, dysregulation, or constitutive activation via a conformational change. RESULTS: PSSM1-affected horse muscle had significantly higher glycogen content than control horse muscle despite no difference in GS expression. GS activity was significantly higher in muscle from homozygous mutants than from heterozygote and control horses, in the absence and presence of the allosteric regulator, glucose 6 phosphate (G6P). Muscle from homozygous mutant horses also had significantly increased GS phosphorylation at sites 2+2a and significantly higher AMPKα1 (an upstream kinase) expression than controls, likely reflecting a physiological attempt to reduce GS enzyme activity. Recombinant mutant GS was highly active with a considerably lower Km for UDP-glucose, in the presence and absence of G6P, when compared to wild type GS, and despite its phosphorylation. CONCLUSIONS: Elevated activity of the mutant enzyme is associated with ineffective regulation via phosphorylation rendering it constitutively active. Modelling suggested that the mutation disrupts a salt bridge that normally stabilises the basal state, shifting the equilibrium to the enzyme's active state. GENERAL SIGNIFICANCE: This study explains the gain of function pathogenesis in this highly prevalent polyglucosan myopathy.

Inhibition of the oxidative metabolism of 3,4-dihydroxyphenylacetaldehyde, a reactive intermediate of dopamine metabolism, by 4-hydroxy-2-nonenal.

Recent evidence indicates a role for oxidative stress and resulting products, e.g. 4-hydroxy-2-nonenal (4HNE) in the pathogenesis of Parkinson's disease (PD). 4HNE is a known inhibitor of mitochondrial aldehyde dehydrogenase (ALDH2), an enzyme very important to the dopamine (DA) metabolic pathway. DA undergoes monoamine oxidase-catalyzed oxidative deamination to 3,4-dihydroxyphenylacetaldehyde (DOPAL), which is metabolized primarily to 3,4-dihydroxyphenylacetic acid (DOPAC) via ALDH2. The biotransformation of DOPAL is critical as previous studies have demonstrated this DA-derived aldehyde to be a reactive electrophile and toxic to dopaminergic cells. Therefore, 4HNE produced via oxidative stress may inhibit ALDH2-mediated oxidation of the endogenous neurotoxin DOPAL. To test this hypothesis, ALDH2 in various model systems was treated with 4HNE and activity toward DOPAL measured. Incubation of human recombinant ALDH2 with 4HNE (1.5-30 microM) yielded inhibition of activity toward DOPAL. Furthermore, ALDH2 in rat brain mitochondrial lysate as well as isolated rat brain mitochondria was also sensitive to the lipid peroxidation product at low micromolar, as evident by a decrease in the rate of DOPAL to DOPAC conversion measured using HPLC. Taken together, these data indicate that 4HNE at low micromolar inhibits mitochondrial biotransformation of DOPAL to DOPAC, and generation of the lipid peroxidation product may represent a mechanism yielding aberrant levels of DOPAL, thus linking oxidative stress to the uncontrolled production of an endogenous neurotoxin relevant to PD.

Structure of mitochondrial aldehyde dehydrogenase: the genetic component of ethanol aversion.

BACKGROUND: The single genetic factor most strongly correlated with reduced alcohol consumption and incidence of alcoholism is a naturally occurring variant of mitochondrial aldehyde dehydrogenase (ALDH2). This variant contains a glutamate to lysine substitution at position 487 (E487K). The E487K variant of ALDH2 is found in approximately 50% of the Asian population, and is associated with a phenotypic loss of ALDH2 activity in both heterozygotes and homozygotes. ALDH2-deficient individuals exhibit an averse response to ethanol consumption, which is probably caused by elevated levels of blood acetaldehyde. The structure of ALDH2 is important for the elucidation of its catalytic mechanism, to gain a clear understanding of the contribution of ALDH2 to the genetic component of alcoholism and for the development of specific ALDH2 inhibitors as potential drugs for use in the treatment of alcoholism. RESULTS: The X-ray structure of bovine ALDH2 has been solved to 2.65 A in its free form and to 2.75 A in a complex with NAD+. The enzyme structure contains three domains; two dinucleotide-binding domains and a small three-stranded beta-sheet domain, which is involved in subunit interactions in this tetrameric enzyme. The E487K mutation occurs in this small oligomerization domain and is located at a key interface between subunits immediately below the active site of another monomer. The active site of ALDH2 is divided into two halves by the nicotinamide ring of NAD+. Adjacent to the A-side (Pro-R) of the nicotinamide ring is a cluster of three cysteines (Cys301, Cys302 and Cys303) and adjacent to the B-side (Pro-S) are Thr244, Glu268, Glu476 and an ordered water molecule bound to Thr244 and Glu476. CONCLUSIONS: Although there is a recognizable Rossmann-type fold, the coenzyme-binding region of ALDH2 binds NAD+ in a manner not seen in other NAD+-binding enzymes. The positions of the residues near the nicotinamide ring of NAD+ suggest a chemical mechanism whereby Glu268 functions as a general base through a bound water molecule. The sidechain amide nitrogen of Asn169 and the peptide nitrogen of Cys302 are in position to stabilize the oxyanion present in the tetrahedral transition state prior to hydride transfer. The functional importance of residue Glu487 now appears to be due to indirect interactions of this residue with the substrate-binding site via Arg264 and Arg475.

Crystallization and preliminary X-ray investigation of bovine liver mitochondrial aldehyde dehydrogenase.

Aldehyde dehydrogenase from bovine liver mitochondria has been crystallized using the sitting drop method of vapor diffusion at 22 degrees C. The crystals formed from solutions containing, 40 mM-sodium citrate, 1 mM-NAD+ and 21% to 24% polyethylene glycol 3400 (pH 5.3 to 5.5). X-ray diffraction data collected from these crystals indicate that the crystals belong to the orthorhombic space group P2(1)2(1)2(1) with cell dimensions of a = 153.7 A, b = 159.37 A and c = 101.45 A. The crystals diffract to at least 2.9 A and a tetramer may comprise the asymmetric unit.

Structure of human chi chi alcohol dehydrogenase: a glutathione-dependent formaldehyde dehydrogenase.

The crystal structure of the human class III chi chi alcohol dehydrogenase (ADH) in a binary complex with NAD+(gamma) was solved to 2.7 A resolution by molecular replacement with human class I beta1 beta1 ADH. chi chi ADH catalyzes the oxidation of long-chain alcohols such as omega-hydroxy fatty acids as well as S-hydroxymethyl-glutathione, a spontaneous adduct between formaldehyde and glutathione. There are two subunits per asymmetric unit in the chi chi ADH structure. Both subunits display a semi-open conformation of the catalytic domain. This conformation is half-way between the open and closed conformations described for the horse EE ADH enzyme. The semi-open conformation and key changes in elements of secondary structure provide a structural basis for the ability of chi chi ADH to bind S-hydroxymethyl-glutathione and 10-hydroxydecanoate. Direct coordination of the catalytic zinc ion by Glu68 creates a novel environment for the catalytic zinc ion in chi chi ADH. This new configuration of the catalytic zinc is similar to an intermediate for horse EE ADH proposed through theoretical computations and is consistent with the spectroscopic data of the Co(II)-substituted chi chi enzyme. The position for residue His47 in the chi chi ADH structure suggests His47 may function both as a catalytic base for proton transfer and in the binding of the adenosine phosphate of NAD(H). Modeling of substrate binding to this enzyme structure is consistent with prior mutagenesis data which showed that both Asp57 and Arg115 contribute to glutathione binding and that Arg115 contributes to the binding of omega-hydroxy fatty acids and identifies additional residues which may contribute to substrate binding.

Three-dimensional structure of mammalian casein kinase I: molecular basis for phosphate recognition.

The three-dimensional structure for the catalytic region of the mammalian protein kinase, casein kinase I delta (CKI delta), has been solved by X-ray crystallography to a resolution of 2.3 A. A truncation mutant of CKI delta lacking the C-terminal autoinhibitory region was expressed in Escherichia coli, purified, and crystallized. The structure was solved by molecular replacement using the crystal structure of the catalytic domain of a CKI homolog from Schizosaccharomyces pombe, Cki1. A tungstate derivative confirmed the initial molecular replacement solution and identified an anion binding site which may contribute to the unique substrate specificity of CKI. Like other protein kinases, the catalytic domain of CKI is composed of two lobes with a cleft between them for binding ATP. Comparison of the mammalian and yeast CKI structures suggests that a rotation of the N-terminal domain occurs upon ATP binding. This domain motion is similar, but not identical, to that observed in cAMP-dependent protein kinase upon binding ATP. Although Cki1 has many similarities to CKI delta over the catalytic domain, these two forms of CKI likely perform different functions in vivo. Relating the primary sequences of other CKI enzymes to the three-dimensional architecture of CKI delta reveals a catalytic face that is especially conserved among the subset of CKI family members associated with the regulation of DNA repair.

Structures of three human beta alcohol dehydrogenase variants. Correlations with their functional differences.

The three-dimensional structures of three variants of human beta alcohol dehydrogenase have been determined to 2.5 A resolution. These three structures differ only in the amino acid at position 47 and the molecules occupying the alcohol binding site. Human beta 1 alcohol dehydrogenase has an Arg at position 47 and was crystallized in a complex with NAD(H) and cyclohexanol. A naturally occurring variant of beta 1 alcohol dehydrogenase, found in approximately 50% of the Asian population, possesses a His at position 47 (beta 2 or beta 47H) and was crystallized in a complex with NAD+ and the inhibitor 4-iodopyrazole. A site-directed mutant of beta 1 alcohol dehydrogenase in which a Gly is substituted for Arg47 (beta 47G) was crystallized in a complex with NAD+. By comparing both the common and unique features of these structures, it is clear that position 47 contributes significantly to the strength of protein-coenzyme interactions. The substitution of Arg47 by His produces an enzyme with a 100-fold lower affinity for coenzyme, but creates no large changes in the enzyme structure. The substitution of Arg47 by Gly produces an enzyme with coenzyme binding characteristics more similar to the wild-type enzyme than to the enzyme with His at position 47, but the structure of the Gly47 variant exhibits differences in and around the coenzyme binding site. These changes involve a rigid-body rotation of the catalytic domain towards the coenzyme domain by approximately 0.8 degrees and local rearrangements of amino acid side-chains, such as a 1.0 A movement of Lys228, relative to the beta 1 enzyme. These structural alterations may compensate for the loss of coenzyme interactions contributed by Arg47 and can explain the high affinity of the Gly47 variant for coenzyme.

ß-Catenin-regulated ALDH1A1 is a target in ovarian cancer spheroids.

Cancer cells form three-dimensional (3D) multicellular aggregates (or spheroids) under non-adherent culture conditions. In ovarian cancer (OC), spheroids serve as a vehicle for cancer cell dissemination in the peritoneal cavity, protecting cells from environmental stress-induced anoikis. To identify new targetable molecules in OC spheroids, we investigated gene expression profiles and networks upregulated in 3D vs traditional monolayer culture conditions. We identified ALDH1A1, a cancer stem cell marker as being overexpressed in OC spheroids and directly connected to key elements of the ß-catenin pathway. ß-Catenin function and ALDH1A1 expression were increased in OC spheroids vs monolayers and in successive spheroid generations, suggesting that 3D aggregates are enriched in cells with stem cell characteristics. ß-Catenin knockdown decreased ALDH1A1 expression levels and ß-catenin co-immunoprecipitated with the ALDH1A1 promoter, suggesting that ALDH1A1 is a direct ß-catenin target. Both short interfering RNA-mediated ß-catenin knockdown and A37 ((ethyl-2-((4-oxo-3-(3-(pryrrolidin-1-yl)propyl)-3,4-dihydrobenzo [4,5]thioeno [3,2-d]pyrimidin-2-yl)thio)acetate)), a novel ALDH1A1 small-molecule enzymatic inhibitor described here for the first time, disrupted OC spheroid formation and cell viability (P<0.001). ß-Catenin knockdown blocked tumor growth and peritoneal metastasis in an OC xenograft model. These data strongly support the role of ß-catenin-regulated ALDH1A1 in the maintenance of OC spheroids and propose new ALDH1A1 inhibitors targeting this cell population.

Methionine-141 directly influences the binding of 4-methylpyrazole in human sigma sigma alcohol dehydrogenase.

Pyrazole and its 4-alkyl substituted derivatives are potent inhibitors for many alcohol dehydrogenases. However, the human sigma sigma isoenzyme exhibits a 580-fold lower affinity for 4-methylpyrazole than does the human beta1beta1 isoenzyme, with which it shares 69% sequence identity. In this study, structural and kinetic studies were utilized in an effort to identify key structural features that affect the binding of 4-methylpyrazole in human alcohol dehydrogenase isoenzymes. We have extended the resolution of the human sigma sigma alcohol dehydrogenase (ADH) isoenzyme to 2.5 A resolution. Comparison of this structure to the human beta1beta1 isoenzyme structure indicated that the side-chain position for Met141 in sigma sigma ADH might interfere with 4-methylpyrazole binding. Mutation of Met141 in sigma sigma ADH to Leu (sigma141L) lowers the Ki for 4-methylpyrazole from 350 to 10 microM, while having a much smaller effect on the Ki for pyrazole. Thus, the mutagenesis results show that the residue at position 141, which lines the substrate-binding pocket at a position close to the methyl group of 4-methylpyrazole, directly affects the binding of the inhibitor. To rule out nonspecific structural changes due to the mutation, the X-ray structure of the sigma141L mutant enzyme was determined to 2.4 A resolution. The three-dimensional structure of the mutant enzyme is identical to the wild-type enzyme, with the exception of the residue at position 141. Thus, the differences in 4-methylpyrazole binding between the mutant and wild-type sigma sigma ADH isoenzymes can be completely ascribed to the local changes in the topology of the substrate binding site, and provides an explanation for the class-specific differences in 4-methylpyrazole binding to the human ADH isoenzymes.

Human liver mitochondrial aldehyde dehydrogenase: three-dimensional structure and the restoration of solubility and activity of chimeric forms.

Human liver cytosolic and mitochondrial isozymes of aldehyde dehydrogenase share 70% sequence identity. However, the first 21 residues are not conserved between the human isozymes (15% identity). The three-dimensional structures of the beef mitochondrial and sheep cytosolic forms have virtually identical three-dimensional structures. Here, we solved the structure of the human mitochondrial enzyme and found it to be identical to the beef enzyme. The first 21 residues are found on the surface of the enzyme and make no contact with other subunits in the tetramer. A pair of chimeric enzymes between the human isozymes was made. Each chimera had the first 21 residues from one isozyme and the remaining 479 from the other. When the first 21 residues were from the mitochondrial isozyme, an enzyme with cytosolic-like properties was produced. The other was expressed but was insoluble. It was possible to restore solubility and activity to the chimera that had the first 21 cytosolic residues fused to the mitochondrial ones by making point mutations to residues at the N-terminal end. When residue 19 was changed from tyrosine to a cysteine, the residue found in the mitochondrial form, an active enzyme could be made though the Km for NAD+ was 35 times higher than the native mitochondrial isozyme and the specific activity was reduced by 75%. This residue interacts with residue 203, a nonconserved, nonactive site residue. A mutation of residue 18, which also interacts with 203, restored solubility, but not activity. Mutation to residue 15, which interacts with 104, also restored solubility but not activity. It appears that to have a soluble or active enzyme a favorable interaction must occur between a residue in a surface loop and a residue elsewhere in the molecule even though neither make contact with the active site region of the enzyme.

Three-dimensional structures of the three human class I alcohol dehydrogenases.

In contrast with other animal species, humans possess three distinct genes for class I alcohol dehydrogenase and show polymorphic variation in the ADH1B and ADH1C genes. The three class I alcohol dehydrogenase isoenzymes share approximately 93% sequence identity but differ in their substrate specificity and their developmental expression. We report here the first three-dimensional structures for the ADH1A and ADH1C*2 gene products at 2.5 and 2.0 A, respectively, and the structure of the ADH1B*1 gene product in a binary complex with cofactor at 2.2 A. Not surprisingly, the overall structure of each isoenzyme is highly similar to the others. However, the substitution of Gly for Arg at position 47 in the ADH1A isoenzyme promotes a greater extent of domain closure in the ADH1A isoenzyme, whereas substitution at position 271 may account for the lower turnover rate for the ADH1C*2 isoenzyme relative to its polymorphic variant, ADH1C*1. The substrate-binding pockets of each isoenzyme possess a unique topology that dictates each isoenzyme's distinct but overlapping substrate preferences. ADH1*B1 has the most restrictive substrate-binding site near the catalytic zinc atom, whereas both ADH1A and ADH1C*2 possess amino acid substitutions that correlate with their better efficiency for the oxidation of secondary alcohols. These structures describe the nature of their individual substrate-binding pockets and will improve our understanding of how the metabolism of beverage ethanol affects the normal metabolic processes performed by these isoenzymes.

Tryptophan fluorescence quenching by alkaline pH and ternary complex formation in human beta 1 beta 1 and horse EE alcohol dehydrogenases.

The horse EE and human beta 1 beta 1 alcohol dehydrogenase isoenzymes have almost identical protein backbone folding patterns and contain 2 tryptophans per subunit (Trp-15 and Trp-314). Tyr-286, which had been proposed to quench the fluorescence of Trp-314 by resonance energy transfer at alkaline pH in EE, is substituted by Cys in beta 1 beta 1. The proposed role of Tyr-286 in pH-dependent quenching of EE is confirmed by our observation that tryptophan fluorescence of beta 1 beta 1 is not substantially quenched at alkaline pH. Tyr-286 had also been implicated in the quenching of Trp-314 upon formation of the EE-NAD(+)-trifluoroethanol ternary complex. However, beta 1 beta 1 exhibits the same extent of tryptophan fluorescence quenching as EE upon complexation, which strongly suggests that Tyr-286 is not involved in ternary complex quenching.

Different mutations in the same codon of the proteolipid protein gene, PLP, may help in correlating genotype with phenotype in Pelizaeus-Merzbacher disease/X-linked spastic paraplegia (PMD/SPG2).

Pelizaeus-Merzbacher disease/X-linked spastic paraplegia (PMD/SPG2) comprises a spectrum of diseases that range from severe to quite mild. The reasons for the variation in severity are not obvious, but suggested explanations include the extent of disruption of the transmembrane portion of the proteolipid protein caused by certain amino acid substitutions and interference with the trafficking of the PLP molecule in oligodendrocytes. Four codons in which substitution of more than one amino acid has occurred are available for examination of clinical and potential structural manifestations: Valine165 to either glutamate or glycine, leucine 045 to either proline or arginine, aspartate 202 to asparagine or histidine, and leucine 223 to isoleucine or proline. Three of these mutations, Val165Gly, Leu045Pro, and Leu223Ile have not been described previously in humans. The altered amino acids appear in the A-B loop, C helix, and C-D loop, respectively. We describe clinically patients with the mutations T494G (Val165Gly), T134C (Leu045Pro), and C667A (Leu223Ile). We discuss also the previously reported mutations Asp202Asn and Asp202His. We have calculated the changes in hydrophobicity of short sequences surrounding some of these amino acids and compared the probable results of the changes in transmembrane structure of the proteolipid protein for the various mutations with the clinical data available on the patients. While the Val165Glu mutation, which is expected to produce disruption of a transmembrane loop of the protein, produces more severe disease than does Val165Gly, no particular correlation with hydrophobicity is found for the other mutations. As these are not in transmembrane domains, other factors such as intracellular transport or interaction between protein chains during myelin formation are probably at work.

Studies on the regulation of the mitochondrial alpha-ketoacid dehydrogenase complexes and their kinases.

Five mitochondrial protein kinases, all members of a new family of protein kinases, have now been identified, cloned, expressed as recombinant proteins, and partially characterized with respect to catalytic and regulatory properties. Four members of this unique family of eukaryotic protein kinases correspond to pyruvate dehydrogenase kinase isozymes which regulate the activity of the pyruvate dehydrogenase complex, an important regulatory enzyme at the interface between glycolysis and the citric acid cycle. The fifth member of this family corresponds to the branched-chain alpha-ketoacid dehydrogenase kinase, an enzyme responsible for phosphorylation and inactivation of the branched-chain alpha-ketoacid dehydrogenase complex, the most important regulatory enzyme in the pathway for the disposal of branched-chain amino acids. At least three long-term control mechanisms have evolved to conserve branched chain amino acids for protein synthesis during periods of dietary protein insufficiency. Increased expression of the branched-chain alpha-ketoacid dehydrogenase kinase is perhaps the most important because this leads to phosphorylation and nearly complete inactivation of the liver branched-chain alpha-ketoacid dehydrogenase complex. Decreased amounts of the liver branched-chain alpha-ketoacid dehydrogenase complex secondary to a decrease in liver mitochondria also decrease the liver's capacity for branched-chain keto acid oxidation. Finally, the number of E1 subunits of the branched-chain alpha-ketoacid dehydrogenase complex is reduced to less than a full complement of 12 heterotetramers per complex in the liver of protein-starved rats. Since the E1 component is rate-limiting for activity and also the component of the complex inhibited by phosphorylation, this decrease in number further limits overall enzyme activity and makes the complex more sensitive to regulation by phosphorylation in this nutritional state. The branched-chain alpha-ketoacid dehydrogenase kinase phosphorylates serine 293 of the E1 alpha subunit of the branched-chain alpha-ketoacid dehydrogenase complex. Site-directed mutagenesis of amino acid residues surrounding serine 293 reveals that arginine 288, histidine 292 and aspartate 296 are critical to dehydrogenase activity, that histidine 292 is critical to binding the coenzyme thiamine pyrophosphate, and that serine 293 exists at or in close proximity to the active site of the dehydrogenase. Alanine scanning mutagenesis of residues in the immediate vicinity of the phosphorylation site (serine 293) indicates that only arginine 288 is required for recognition of serine 293 as a phosphorylation site by the branched-chain alpha-ketoacid dehydrogenase kinase. Phosphorylation appears to inhibit dehydrogenase activity by introducing a negative charge directly into the active site pocket of the E1 dehydrogenase component of the branched-chain alpha-ketoacid dehydrogenase complex. A model based on the X-ray crystal structure of transketolase is being used to predict residues involved in thiamine pyrophosphate binding and to help visualize how phosphorylation within the channel leading to the reactive carbon of thiamine pyrophosphate inhibits catalytic activity. The isoenzymes of pyruvate dehydrogenase kinase differ greatly in terms of their specific activities, kinetic parameters and regulatory properties. Chemically-induced diabetes in the rat induces significant changes in the pyruvate dehydrogenase kinase isoenzyme 2 in liver. Preliminary findings suggest hormonal control of the activity state of the pyruvate dehydrogenase complex may involves tissue specific induced changes in expression of the pyruvate dehydrogenase kinase isoenzymes.

Order and disorder in mitochondrial aldehyde dehydrogenase.

One of the most notable and currently unexplained features of the mitochondrial form of aldehyde dehydrogenase is its property of half-of-the-sites reactivity. An appropriate description of this phenomenon can be to consider this as the extreme example of negative cooperativity. This implies, therefore, that a pathway of communication must exist between active sites in order to convey the structural consequences of ligand binding. Data from four different structures of human ALDH2 collected during the past 2 years may shed some light on one possible pathway for the propagation of structural information. We recently published a 2.6 A structure of a binary complex between ALDH2 and NAD(+) in which the predominant conformation of the cofactor differed between different subunits in the structure. We now have three unpublished structures, a wild-type apo-enzyme structure at 2.25 A resolution, a wild-type structure complexed with NADH at 2.45 A resolution, and a site-directed mutant of ALDH2 where Arg475 is mutated to Gln, as an apo-enzyme to 2.75 A resolution. A detailed comparison of their structures reveals that a disorder-to-order transition occurs upon coenzyme binding in the area immediately surrounding the adenosine-binding site (residues 224-233 and 246-262). These residues correspond to the two helices that surround the adenine ring of the cofactor. Since the helix comprised of residues 246-262 contacts its dimer related helix across the subunit interface, this could induce as of yet unidentified subtle changes in structure that impair productive binding of the cofactor in the second subunit. The unique characteristics and three-dimensional structure of the R475Q variant of ALDH2 supports a role in subunit communication for these residues. This mutated enzyme displays positive cooperativity for cofactor binding. The structure of the apo-enzyme shows that the average thermal parameters for the residues involved in adenosine binding are drastically elevated as is a stretch of amino acids surrounding the site of mutation (residues 471-480). We hypothesize that cofactor binding displays a Hill coefficient of approximately 2 because binding of coenzyme to one subunit in a dimer orders the residues responsible for cofactor binding in the second, thus promoting binding. The difference between these alterations being positively versus negatively cooperative is likely related to the magnitude of the structural changes. Further work is in progress to confirm this hypothesis as it may shed light on the dominant effects of the E487K allelic variant, since Glu487 interacts with Arg475.