Phosphorylation of Thr9 Affects the Folding Landscape of the N-Terminal Segment of Human AGT Enhancing Protein Aggregation of Disease-Causing Mutants.

The mutations G170R and I244T are the most common disease cause in primary hyperoxaluria type I (PH1). These mutations cause the misfolding of the AGT protein in the minor allele AGT-LM that contains the P11L polymorphism, which may affect the folding of the N-terminal segment (NTT-AGT). The NTT-AGT is phosphorylated at T9, although the role of this event in PH1 is unknown. In this work, phosphorylation of T9 was mimicked by introducing the T9E mutation in the NTT-AGT peptide and the full-length protein. The NTT-AGT conformational landscape was studied by circular dichroism, NMR, and statistical mechanical methods. Functional and stability effects on the full-length AGT protein were characterized by spectroscopic methods. The T9E and P11L mutations together reshaped the conformational landscape of the isolated NTT-AGT peptide by stabilizing ordered conformations. In the context of the full-length AGT protein, the T9E mutation had no effect on the overall AGT function or conformation, but enhanced aggregation of the minor allele (LM) protein and synergized with the mutations G170R and I244T. Our findings indicate that phosphorylation of T9 may affect the conformation of the NTT-AGT and synergize with PH1-causing mutations to promote aggregation in a genotype-specific manner. Phosphorylation should be considered a novel regulatory mechanism in PH1 pathogenesis.

A single evolutionarily divergent mutation determines the different FAD-binding affinities of human and rat NQO1 due to site-specific phosphorylation.

The phosphomimetic mutation S82D in the cancer-associated, FAD-dependent human NADP(H):quinone oxidoreductase 1 (hNQO1) causes a decrease in flavin-adenine dinucleotide-binding affinity and intracellular stability. We test in this work whether the evolutionarily recent neutral mutation R80H in the vicinity of S82 may alter the strong functional effects of S82 phosphorylation through electrostatic interactions. We show using biophysical and bioinformatic analyses that the reverse mutation H80R prevents the effects of S82D phosphorylation on hNQO1 by modulating the local stability. Consistently, in rat NQO1 (rNQO1) which contains R80, the effects of phosphorylation were milder, resembling the behaviour found in hNQO1 when this residue was humanized in rNQO1 (by the R80H mutation). Thus, apparently neutral and evolutionarily divergent mutations may determine the functional response of mammalian orthologues towards phosphorylation.

Structural basis of the pleiotropic and specific phenotypic consequences of missense mutations in the multifunctional NAD(P)H:quinone oxidoreductase 1 and their pharmacological rescue.

The multifunctional nature of human flavoproteins is critically linked to their ability to populate multiple conformational states. Ligand binding, post-translational modifications and disease-associated mutations can reshape this functional landscape, although the structure-function relationships of these effects are not well understood. Herein, we characterized the structural and functional consequences of two mutations (the cancer-associated P187S and the phosphomimetic S82D) on different ligation states which are relevant to flavin binding, intracellular stability and catalysis of the disease-associated NQO1 flavoprotein. We found that these mutations affected the stability locally and their effects propagated differently through the protein structure depending both on the nature of the mutation and the ligand bound, showing directional preference from the mutated site and leading to specific phenotypic manifestations in different functional traits (FAD binding, catalysis and inhibition, intracellular stability and pharmacological response to ligands). Our study thus supports that pleitropic effects of disease-causing mutations and phosphorylation events on human flavoproteins may be caused by long-range structural propagation of stability effects to different functional sites that depend on the ligation-state and site-specific perturbations. Our approach can be of general application to investigate these pleiotropic effects at the flavoproteome scale in the absence of high-resolution structural models.

NQO1: A target for the treatment of cancer and neurological diseases, and a model to understand loss of function disease mechanisms.

NAD(P)H quinone oxidoreductase 1 (NQO1) is a multi-functional protein that catalyses the reduction of quinones (and other molecules), thus playing roles in xenobiotic detoxification and redox balance, and also has roles in stabilising apoptosis regulators such as p53. The structure and enzymology of NQO1 is well-characterised, showing a substituted enzyme mechanism in which NAD(P)H binds first and reduces an FAD cofactor in the active site, assisted by a charge relay system involving Tyr-155 and His-161. Protein dynamics play important role in physio-pathological aspects of this protein. NQO1 is a good target to treat cancer due to its overexpression in cancer cells. A polymorphic form of NQO1 (p.P187S) is associated with increased cancer risk and certain neurological disorders (such as multiple sclerosis and Alzheimer´s disease), possibly due to its roles in the antioxidant defence. p.P187S has greatly reduced FAD affinity and stability, due to destabilization of the flavin binding site and the C-terminal domain, which leading to reduced activity and enhanced degradation. Suppressor mutations partially restore the activity of p.P187S by local stabilization of these regions, and showing long-range allosteric communication within the protein. Consequently, the correction of NQO1 misfolding by pharmacological chaperones is a viable strategy, which may be useful to treat cancer and some neurological conditions, targeting structural spots linked to specific disease-mechanisms. Thus, NQO1 emerges as a good model to investigate loss of function mechanisms in genetic diseases as well as to improve strategies to discriminate between neutral and pathogenic variants in genome-wide sequencing studies.

Structural and functional insights on the roles of molecular chaperones in the mistargeting and aggregation phenotypes associated with primary hyperoxaluria type I.

To carry out their biological function in cells, proteins must be folded and targeted to the appropriate subcellular location. These processes are controlled by a vast collection of interacting proteins collectively known as the protein homeostasis network, in which molecular chaperones play a prominent role. Protein homeostasis can be impaired by inherited mutations leading to genetic diseases. In this chapter, we focus on a particular disease, primary hyperoxaluria type 1 (PH1), in which disease-associated mutations exacerbate protein aggregation in the cell and mistarget the peroxisomal alanine:glyoxylate aminotransferase (AGT) protein to mitochondria, in part due to native state destabilization and enhanced interaction with Hsp60, 70 and 90 chaperone systems. After a general introduction of molecular chaperones and PH1, we review our current knowledge on the structural and energetic features of PH1-causing mutants that lead to these particular pathogenic mechanisms. From this perspective, and in the context of the key role of molecular chaperones in PH1 pathogenesis, we present and discuss current and future perspectives for pharmacological treatments for this disease.

Insight into the specificity and severity of pathogenic mechanisms associated with missense mutations through experimental and structural perturbation analyses.

Most pathogenic missense mutations cause specific molecular phenotypes through protein destabilization. However, how protein destabilization is manifested as a given molecular phenotype is not well understood. We develop here a structural and energetic approach to describe mutational effects on specific traits such as function, regulation, stability, subcellular targeting or aggregation propensity. This approach is tested using large-scale experimental and structural perturbation analyses in over thirty mutations in three different proteins (cancer-associated NQO1, transthyretin related with amyloidosis and AGT linked to primary hyperoxaluria type I) and comprising five very common pathogenic mechanisms (loss-of-function and gain-of-toxic function aggregation, enzyme inactivation, protein mistargeting and accelerated degradation). Our results revealed that the magnitude of destabilizing effects and, particularly, their propagation through the structure to promote disease-associated conformational states largely determine the severity and molecular mechanisms of disease-associated missense mutations. Modulation of the structural perturbation at a mutated site is also shown to cause switches between different molecular phenotypes. When very common disease-associated missense mutations were investigated, we also found that they were not among the most deleterious possible missense mutations at those sites, and required additional contributions from codon bias and effects of CpG sites to explain their high frequency in patients. Our work sheds light on the molecular basis of pathogenic mechanisms and genotype-phenotype relationships, with implications for discriminating between pathogenic and neutral changes within human genome variability from whole genome sequencing studies.

Phosphorylation compromises FAD binding and intracellular stability of wild-type and cancer-associated NQO1: Insights into flavo-proteome stability.

Over a quarter million of protein phosphorylation sites have been identified so far, although the effects of site-specific phosphorylation on protein function and stability, as well as their possible impact in the phenotypic manifestation in genetic diseases are vastly unknown. We investigated here the effects of phosphorylating S82 in human NADP(H):quinone oxidoreductase 1, a representative example of disease-associated flavoprotein in which protein stability is coupled to the intracellular flavin levels. Additionally, the cancer-associated P187S polymorphism causes inactivation and destabilization of the enzyme. By using extensive in vitro and in silico characterization of phosphomimetic S82D mutations, we showed that S82D locally affected the flavin binding site of the wild-type (WT) and P187S proteins thus altering flavin binding affinity, conformational stability and aggregation propensity. Consequently, the phosphomimetic S82D may destabilize the WT protein intracellularly by promoting the formation of the degradation-prone apo-protein. Noteworthy, WT and P187S proteins respond differently to the phosphomimetic mutation in terms of intracellular stability, further supporting differences in molecular recognition of these two variants by the proteasomal degradation pathway. We propose that phosphorylation could have critical consequences on stability and function of human flavoproteins, important for our understanding of genotype-phenotype relationships in their related genetic diseases.

High-Affinity Chemotaxis to Histamine Mediated by the TlpQ Chemoreceptor of the Human Pathogen Pseudomonas aeruginosa.

Histamine is a key biological signaling molecule. It acts as a neurotransmitter in the central and peripheral nervous systems and coordinates local inflammatory responses by modulating the activity of different immune cells. During inflammatory processes, including bacterial infections, neutrophils stimulate the production and release of histamine. Here, we report that the opportunistic human pathogen Pseudomonas aeruginosa exhibits chemotaxis toward histamine. This chemotactic response is mediated by the concerted action of the TlpQ, PctA, and PctC chemoreceptors, which display differing sensitivities to histamine. Low concentrations of histamine were sufficient to activate TlpQ, which binds histamine with an affinity of 639 nM. To explore this binding, we resolved the high-resolution structure of the TlpQ ligand binding domain in complex with histamine. It has an unusually large dCACHE domain and binds histamine through a highly negatively charged pocket at its membrane distal module. Chemotaxis to histamine may play a role in the virulence of P. aeruginosa by recruiting cells at the infection site and consequently modulating the expression of quorum-sensing-dependent virulence genes. TlpQ is the first bacterial histamine receptor to be described and greatly differs from human histamine receptors, indicating that eukaryotes and bacteria have pursued different strategies for histamine recognition.IMPORTANCE Genome analyses indicate that many bacteria possess an elevated number of chemoreceptors, suggesting that these species are able to perform chemotaxis to a wide variety of compounds. The scientific community is now only beginning to explore this diversity and to elucidate the corresponding physiological relevance. The discovery of histamine chemotaxis in the human pathogen Pseudomonas aeruginosa provides insight into tactic movements that occur within the host. Since histamine is released in response to bacterial pathogens, histamine chemotaxis may permit bacterial migration and accumulation at infection sites, potentially modulating, in turn, quorum-sensing-mediated processes and the expression of virulence genes. As a consequence, the modulation of histamine chemotaxis by signal analogues may result in alterations of the bacterial virulence. As the first report of bacterial histamine chemotaxis, this study lays the foundation for the exploration of the physiological relevance of histamine chemotaxis and its role in pathogenicity.

Evolutionary Divergent Suppressor Mutations in Conformational Diseases.

Neutral and adaptive mutations are key players in the evolutionary dynamics of proteins at molecular, cellular and organismal levels. Conversely, largely destabilizing mutations are rarely tolerated by evolution, although their occurrence in diverse human populations has important roles in the pathogenesis of conformational diseases. We have recently proposed that divergence at certain sites from the consensus (amino acid) state during mammalian evolution may have rendered some human proteins more vulnerable towards disease-associated mutations, primarily by decreasing their conformational stability. We herein extend and refine this hypothesis discussing results from phylogenetic and structural analyses, structure-based energy calculations and structure-function studies at molecular and cellular levels. As proof-of-principle, we focus on different mammalian orthologues of the NQO1 (NAD(P)H:quinone oxidoreductase 1) and AGT (alanine:glyoxylate aminotransferase) proteins. We discuss the different loss-of-function pathogenic mechanisms associated with diseases involving the two enzymes, including enzyme inactivation, accelerated degradation, intracellular mistargeting, and aggregation. Last, we take into account the potentially higher robustness of mammalian orthologues containing certain consensus amino acids as suppressors of human disease, and their relation with different intracellular post-translational modifications and protein quality control capacities, to be discussed as sources of phenotypic variability between human and mammalian models of disease and as tools for improving current therapeutic approaches.

Improving the Activity and Stability of Human Galactokinase for Therapeutic and Biotechnological Applications.

Galactokinase catalyses the site- and stereospecific phosphorylation of α-d-galactose. As such it has attracted interest as a biocatalyst for the introduction of phosphate groups into monosaccharides. However, attempts to broaden the substrate range of human galactokinase have generally resulted in substantially reduced activity. The enzyme also has biotechnological potential in enzyme replacement therapy (ERT) for type II galactosaemia. The return-to-consensus approach can be used to identify residues that can be altered to increase protein stability and enzyme activity. This approach identified six residues of potential interest in human galactokinase. Some of the single consensus variants (M60V, D268E, A334S and G373S) increased the catalytic turnover of the enzyme, but none resulted in improved stability. When all six changes were introduced into the protein (M60V/M180V/D268E/A334S/R366Q/G373S), thermal stability was increased. Molecular dynamics simulations suggested that these changes altered the protein's conformation at key sites. The number of salt bridges and hydrogen bonds was also increased. Combining the six consensus variations with Y379W (a variant with greater substrate promiscuity) increased the stability of this variant and its turnover towards some substrates. Thus, the six consensus variants can be used to stabilise catalytically interesting variants of human galactokinase and might also be useful if the protein were to be used in ERT.

Enhanced vulnerability of human proteins towards disease-associated inactivation through divergent evolution.

Human proteins are vulnerable towards disease-associated single amino acid replacements affecting protein stability and function. Interestingly, a few studies have shown that consensus amino acids from mammals or vertebrates can enhance protein stability when incorporated into human proteins. Here, we investigate yet unexplored relationships between the high vulnerability of human proteins towards disease-associated inactivation and recent evolutionary site-specific divergence of stabilizing amino acids. Using phylogenetic, structural and experimental analyses, we show that divergence from the consensus amino acids at several sites during mammalian evolution has caused local protein destabilization in two human proteins linked to disease: cancer-associated NQO1 and alanine:glyoxylate aminotransferase, mutated in primary hyperoxaluria type I. We demonstrate that a single consensus mutation (H80R) acts as a disease suppressor on the most common cancer-associated polymorphism in NQO1 (P187S). The H80R mutation reactivates P187S by enhancing FAD binding affinity through local and dynamic stabilization of its binding site. Furthermore, we show how a second suppressor mutation (E247Q) cooperates with H80R in protecting the P187S polymorphism towards inactivation through long-range allosteric communication within the structural ensemble of the protein. Our results support that recent divergence of consensus amino acids may have occurred with neutral effects on many functional and regulatory traits of wild-type human proteins. However, divergence at certain sites may have increased the propensity of some human proteins towards inactivation due to disease-associated mutations and polymorphisms. Consensus mutations also emerge as a potential strategy to identify structural hot-spots in proteins as targets for pharmacological rescue in loss-of-function genetic diseases.

Caenorhabditis elegans AGXT-1 is a mitochondrial and temperature-adapted ortholog of peroxisomal human AGT1: New insights into between-species divergence in glyoxylate metabolism.

In humans, glyoxylate is an intermediary product of metabolism, whose concentration is finely balanced. Mutations in peroxisomal alanine:glyoxylate aminotransferase (hAGT1) cause primary hyperoxaluria type 1 (PH1), which results in glyoxylate accumulation that is converted to toxic oxalate. In contrast, glyoxylate is used by the nematode Caenorhabditis elegans through a glyoxylate cycle to by-pass the decarboxylation steps of the tricarboxylic acid cycle and thus contributing to energy production and gluconeogenesis from stored lipids. To investigate the differences in glyoxylate metabolism between humans and C. elegans and to determine whether the nematode might be a suitable model for PH1, we have characterized here the predicted nematode ortholog of hAGT1 (AGXT-1) and compared its molecular properties with those of the human enzyme. Both enzymes form active PLP-dependent dimers with high specificity towards alanine and glyoxylate, and display similar three-dimensional structures. Interestingly, AGXT-1 shows 5-fold higher activity towards the alanine/glyoxylate pair than hAGT1. Thermal and chemical stability of AGXT-1 is lower than that of hAGT1, suggesting temperature-adaptation of the nematode enzyme linked to the lower optimal growth temperature of C. elegans. Remarkably, in vivo experiments demonstrate the mitochondrial localization of AGXT-1 in contrast to the peroxisomal compartmentalization of hAGT1. Our results support the view that the different glyoxylate metabolism in the nematode is associated with the divergent molecular properties and subcellular localization of the alanine:glyoxylate aminotransferase activity.

Conformational dynamics is key to understanding loss-of-function of NQO1 cancer-associated polymorphisms and its correction by pharmacological ligands.

Protein dynamics is essential to understand protein function and stability, even though is rarely investigated as the origin of loss-of-function due to genetic variations. Here, we use biochemical, biophysical, cell and computational biology tools to study two loss-of-function and cancer-associated polymorphisms (p.R139W and p.P187S) in human NAD(P)H quinone oxidoreductase 1 (NQO1), a FAD-dependent enzyme which activates cancer pro-drugs and stabilizes several oncosuppressors. We show that p.P187S strongly destabilizes the NQO1 dimer in vitro and increases the flexibility of the C-terminal domain, while a combination of FAD and the inhibitor dicoumarol overcome these alterations. Additionally, changes in global stability due to polymorphisms and ligand binding are linked to the dynamics of the dimer interface, whereas the low activity and affinity for FAD in p.P187S is caused by increased fluctuations at the FAD binding site. Importantly, NQO1 steady-state protein levels in cell cultures correlate primarily with the dynamics of the C-terminal domain, supporting a directional preference in NQO1 proteasomal degradation and the use of ligands binding to this domain to stabilize p.P187S in vivo. In conclusion, protein dynamics are fundamental to understanding loss-of-function in p.P187S, and to develop new pharmacological therapies to rescue this function.

Molecular recognition of PTS-1 cargo proteins by Pex5p: implications for protein mistargeting in primary hyperoxaluria.

Peroxisomal biogenesis and function critically depends on the import of cytosolic proteins carrying a PTS1 sequence into this organelle upon interaction with the peroxin Pex5p. Recent structural studies have provided important insights into the molecular recognition of cargo proteins by Pex5p. Peroxisomal import is a key feature in the pathogenesis of primary hyperoxaluria type 1 (PH1), where alanine:glyoxylate aminotransferase (AGT) undergoes mitochondrial mistargeting in about a third of patients. Here, we study the molecular recognition of PTS1 cargo proteins by Pex5p using oligopeptides and AGT variants bearing different natural PTS1 sequences, and employing an array of biophysical, computational and cell biology techniques. Changes in affinity for Pex5p (spanning over 3-4 orders of magnitude) reflect different thermodynamic signatures, but overall bury similar amounts of molecular surface. Structure/energetic analyses provide information on the contribution of ancillary regions and the conformational changes induced in Pex5p and the PTS1 cargo upon complex formation. Pex5p stability in vitro is enhanced upon cargo binding according to their binding affinities. Moreover, we provide evidence that the rational modulation of the AGT: Pex5p binding affinity might be useful tools to investigate mistargeting and misfolding in PH1 by pulling the folding equilibria towards the native and peroxisomal import competent state.

The lower limits for protein stability and foldability in primary hyperoxaluria type I.

Mutational effects on protein stability and foldability are important to understand conformational diseases and protein evolution. In this work, we perform a comprehensive investigation on the energetic basis underlying mutational effects on the stability of human alanine:glyoxylate aminotransferase (AGT). We study twenty two variants whose kinetic stabilities span over eleven orders of magnitude and are classified into two groups: i) ten naturally-occurring variants, including the most common mutations causing primary hyperoxaluria type I (PH1); and ii) twelve consensus variants obtained by sequence-alignment statistics. We show that AGT dimer stability determines denaturation rates, and mutations modulate stability by changes in the effective thermodynamic stability, the aggregation propensity of partially/globally unfolded states and subtle energetic changes in the rate-limiting denaturation step. In combination with our previous expression analyses in eukaryotic cells, we propose the existence of two lower limits for AGT stability, one linked to optimal folding efficiency (close to the major allele stability) and the other setting a minimal efficiency compatible with glyoxylate detoxification in vivo (close to the minor allele stability). These lower limits could explain the high prevalence of misfolding as a disease mechanism in PH1 and support the use of pharmacological ligands aimed to increase AGT stability as therapies for this disease.

The metastability of human UDP-galactose 4'-epimerase (GALE) is increased by variants associated with type III galactosemia but decreased by substrate and cofactor binding.

Type III galactosemia is an inherited disease caused by mutations which affect the activity of UDP-galactose 4'-epimerase (GALE). We evaluated the impact of four disease-associated variants (p.N34S, p.G90E, p.V94M and p.K161N) on the conformational stability and dynamics of GALE. Thermal denaturation studies showed that wild-type GALE denatures at temperatures close to physiological, and disease-associated mutations often reduce GALE's thermal stability. This denaturation is under kinetic control and results partly from dimer dissociation. The natural ligands, NAD(+) and UDP-glucose, stabilize GALE. Proteolysis studies showed that the natural ligands and disease-associated variations affect local dynamics in the N-terminal region of GALE. Proteolysis kinetics followed a two-step irreversible model in which the intact protein is cleaved at Ala38 forming a long-lived intermediate in the first step. NAD(+) reduces the rate of the first step, increasing the amount of undigested protein whereas UDP-glucose reduces the rate of the second step, increasing accumulation of the intermediate. Disease-associated variants affect these rates and the amounts of protein in each state. Our results also suggest communication between domains in GALE. We hypothesize that, in vivo, concentrations of natural ligands modulate GALE stability and that it should be possible to discover compounds which mimic the stabilising effects of the natural ligands overcoming mutation-induced destabilization.

The consensus-based approach for gene/enzyme replacement therapies and crystallization strategies: the case of human alanine-glyoxylate aminotransferase.

Protein stability is a fundamental issue in biomedical and biotechnological applications of proteins. Among these applications, gene- and enzyme-replacement strategies are promising approaches to treat inherited diseases that may benefit from protein engineering techniques, even though these beneficial effects have been largely unexplored. In the present study we apply a sequence-alignment statistics procedure (consensus-based approach) to improve the activity and stability of the human AGT (alanine-glyoxylate aminotransferase) protein, an enzyme which causes PH1 (primary hyperoxaluria type I) upon mutation. By combining only five consensus mutations, we obtain a variant (AGT-RHEAM) with largely enhanced in vitro thermal and kinetic stability, increased activity, and with no side effects on foldability and peroxisomal targeting in mammalian cells. The structure of AGT-RHEAM reveals changes at the dimer interface and improved electrostatic interactions responsible for increased kinetic stability. Consensus-based variants maintained the overall protein fold, crystallized more easily and improved the expression as soluble proteins in two different systems [AGT and CIPK24 (CBL-interacting serine/threonine-protein kinase) SOS2 (salt-overly-sensitive 2)]. Thus the consensus-based approach also emerges as a simple and generic strategy to increase the crystallization success for hard-to-get protein targets as well as to enhance protein stability and function for biomedical applications.

The role of protein denaturation energetics and molecular chaperones in the aggregation and mistargeting of mutants causing primary hyperoxaluria type I.

Primary hyperoxaluria type I (PH1) is a conformational disease which result in the loss of alanine:glyoxylate aminotransferase (AGT) function. The study of AGT has important implications for protein folding and trafficking because PH1 mutants may cause protein aggregation and mitochondrial mistargeting. We herein describe a multidisciplinary study aimed to understand the molecular basis of protein aggregation and mistargeting in PH1 by studying twelve AGT variants. Expression studies in cell cultures reveal strong protein folding defects in PH1 causing mutants leading to enhanced aggregation, and in two cases, mitochondrial mistargeting. Immunoprecipitation studies in a cell-free system reveal that most mutants enhance the interactions with Hsc70 chaperones along their folding process, while in vitro binding experiments show no changes in the interaction of folded AGT dimers with the peroxisomal receptor Pex5p. Thermal denaturation studies by calorimetry support that PH1 causing mutants often kinetically destabilize the folded apo-protein through significant changes in the denaturation free energy barrier, whereas coenzyme binding overcomes this destabilization. Modeling of the mutations on a 1.9 Å crystal structure suggests that PH1 causing mutants perturb locally the native structure. Our work support that a misbalance between denaturation energetics and interactions with chaperones underlie aggregation and mistargeting in PH1, suggesting that native state stabilizers and protein homeostasis modulators are potential drugs to restore the complex and delicate balance of AGT protein homeostasis in PH1.

Structural and energetic basis of protein kinetic destabilization in human phosphoglycerate kinase 1 deficiency.

Protein kinetic destabilization is a common feature of many human genetic diseases. Human phosphoglycerate kinase 1 (PGK1) deficiency is a rare genetic disease caused by mutations in the PGK1 protein, which often shows reduced kinetic stability. In this work, we have performed an in-depth characterization of the thermal stability of the wild type and four disease-causing mutants (I47N, L89P, E252A, and T378P) of human PGK1. PGK1 thermal denaturation is a process under kinetic control, and it is described well by a two-state irreversible denaturation model. Kinetic analysis of differential scanning calorimetry profiles shows that the disease-causing mutations decrease PGK1 kinetic stability from ~5-fold (E252A) to ~100000-fold (L89P) compared to that of wild-type PGK1, and in some cases, mutant enzymes are denatured on a time scale of a few minutes at physiological temperature. We show that changes in protein kinetic stability are associated with large differences in enthalpic and entropic contributions to denaturation free energy barriers. It is also shown that the denaturation transition state becomes more nativelike in terms of solvent exposure as the protein is destabilized by mutations (Hammond effect). Unfolding experiments with urea further suggest a scenario in which the thermodynamic stability of PGK1 at least partly determines its kinetic stability. ATP and ADP kinetically stabilize PGK1 enzymes, and kinetic stabilization is nucleotide- and mutant-selective. Overall, our data provide insight into the structural and energetic basis underlying the low kinetic stability displayed by some mutants causing human PGK1 deficiency, which may have important implications for the development of native state kinetic stabilizers for the treatment of this disease.