Phosphotransfer Reaction Research Articles

Metal-free cAMP-dependent protein kinase can catalyze phosphoryl transfer.

X-ray structures of several ternary product complexes of the catalytic subunit of cAMP-dependent protein kinase (PKAc) have been determined with no bound metal ions and with Na+ or K+ coordinated at two metal-binding sites. The metal-free PKAc and the enzyme with alkali metals were able to facilitate the phosphoryl transfer reaction. In all studied complexes, the ATP and the substrate peptide (SP20) were modified into the products ADP and the phosphorylated peptide. The products of the phosphotransfer reaction were also found when ATP-γS, a nonhydrolyzable ATP analogue, reacted with SP20 in the PKAc active site containing no metals. Single turnover enzyme kinetics measurements utilizing 32P-labeled ATP confirmed the phosphotransferase activity of the enzyme in the absence of metal ions and in the presence of alkali metals. In addition, the structure of the apo-PKAc binary complex with SP20 suggests that the sequence of binding events may become ordered in a metal-free environment, with SP20 binding first to prime the enzyme for subsequent ATP binding. Comparison of these structures reveals conformational and hydrogen bonding changes that might be important for the mechanism of catalysis.

Profiling of protein thiophosphorylation by Phos‐tag affinity electrophoresis: Evaluation of adenosine 5′‐O‐(3‐thiotriphosphate) as a phosphoryl donor in protein kinase reactions

Adenosine 5'-O-(3-thiotriphosphate) (ATPγS) has been widely used as a phosphoryl donor to trace protein kinase activities. However, the question remains whether particular kinases accept ATPγS as readily as they accept natural ATP. We investigated the characteristics of several kinase reactions in the presence of ATPγS by using Phos-tag affinity electrophoresis. The Phos-tag gel permitted quantitative analysis of thiophosphorylated proteins produced by kinase reactions in vitro and it identified differences in the efficiencies of utilization of ATPγS and ATP in these reactions. Using the method, we evaluated the utility of ATPγS as a phosphoryl donor in studies on bacterial two-component systems. Histidine kinases accepted ATPγS as readily as they accepted ATP in autophosphorylation reactions. However, downstream phosphotransfer reactions with ATPγS were markedly slower than the corresponding reactions with ATP. In an analysis of the sluggish thiophosphate transfer, we found that detergent-denatured thiophosphorylated histidine kinases gradually hydrolyzed at the P-N bond, even at neutral pH, during incubation for 24 h, whereas the native form of the thiophosphorylated enzymes were much more stable. Profiling of protein thiophosphorylation by using Phos-tag affinity electrophoresis might provide new insights into the characteristics of various types of kinase reactions with ATPγS.

Molecular Dynamics Simulations of the Catalytic Subunit of Protein Kinase a Reveal New Insight into the Catalytic Process

Protein kinases chemically add phosphate groups to target proteins. Phosphorylation usually results in a functional change of the target protein, and serves as a regulatory mechanism to elicit a response to a stimulus at the cellular or organism level. Protein kinase A (PKA) was first characterized in 1968 and is considered to be the prototype for the entire kinome. The inactive form of PKA is a heterotetramer of two catalytic subunits (PKAc) that are bound to each member of a regulatory subunit (PKAr) dimer. PKA activates when two cyclic-AMPs bind to each of the PKAr, which releases the PKAc to perform the phosphorylation reaction on a polypeptide substrate. PKAc has two lobes that flank the active site: a small N-terminal lobe that is primarily associated with binding and positioning ATP, and a large lobe that provides a docking surface for substrates or inhibitor proteins. PKAc has three major conformational states: open, intermediate and closed. Despite its early discovery, its activation and deactivation is still not fully understood. Although NMR spin relaxation experiments have probed local dynamics, the movements of the PKAc domains, that may be related to its biological activity, have not been directly examined. To gain new insight into the role that PKAc dynamics plays in the phosphotransfer reaction, we performed microsecond long molecular dynamics simulations of apo-PKAc, PKAc-ATP (intermediate) and PKAc-ATP-substrate peptide (closed, reactive) and PKAc-ATP-inhibitor peptide (closed, nonreactive) complexes. By applying a network-based analysis method, we found conformational communities for PKAc in the various states that made it possible to identify critical residues and structural transitions involved in the catalytic process.

An Allosteric Signaling Pathway of Human 3-Phosphoglycerate Kinase

3-Phosphoglycerate kinase (PGK) catalyzes the phospho-transfer reaction between 1,3-bisphosphoglycerate and ADP. It is a two domain enzyme, with the two substrates bound to the two separate domains. In order to perform its function the enzyme has to undergo a large conformational change involving a hinge bending to bring the substrates into close proximity. The allosteric pathway from the open non-reactive state of PGK to the closed reactive state as triggered by substrate binding has only been partially uncovered by experimental studies. Here we describe a complete allosteric pathway, which connects the substrate binding sites to the interdomain hinge region using Molecular Dynamics simulations combined with Force Distribution Analysis (FDA). While previously identified key residues involved in PGK domain closure are part of this pathway, we here fill the numerous gaps in the pathway by identifying newly uncovered residues and interesting candidates for future mutational studies.

The Role of Soluble 5'.-Nucleotidases in the Conversion of Nucleotide Analogs: Metabolic and Therapeutic Aspects

A variety of anti-proliferative drugs is based on the structure of purine or pyrimidine nucleosides. These compounds, after phosphorylation, act as analogs of natural nucleotides. In vivo they are recognized by enzymes that transform them either into anti-metabolites targeted to the synthesis of DNA or RNA, or to inactive products of detoxification. 5'-Nucleotidases of different specificity and cellular localization can either remove the phosphate residue from the 5'- position of (deoxy) nucleotide or transfer it from nucleoside monophosphate onto other nucleosides. Such a nucleoside phosphotransferase activity also works with analogs of canonical nucleotides and nucleosides. The majority of nucleoside analogs is metabolized by intracellular cytoplasmic or mitochondrial 5'-nucleotidases and only few reactions proceed on the cell surface. This review summarizes our knowledge of cytoplasmic and mitochondrial forms of 5'-nucleotidases and focuses on their ability to dephosphorylate different analogs of canonical nucleoside 5'-monophosphates. The involvement of 5'-nucleotidases in the phosphotransfer reaction with some nucleoside analogs has been also presented. The importance of the reactions catalyzed by 5'-nucleotidases in clinical resistance to nucleoside-based drugs used in the treatment of cancer or viral diseases, as well as in activation of pro-drugs has been highlighted.

Unique Kinase Catalytic Mechanism of AceK with a Single Magnesium Ion

Isocitrate dehydrogenase kinase/phosphatase (AceK) is the founding member of the protein phosphorylation system in prokaryotes. Based on the novel and unique structural characteristics of AceK recently uncovered, we sought to understand its kinase reaction mechanism, along with other features involved in the phosphotransfer process. Herein we report density functional theory QM calculations of the mechanism of the phosphotransfer reaction catalysed by AceK. The transition states located by the QM calculations indicate that the phosphorylation reaction, catalysed by AceK, follows a dissociative mechanism with Asp457 serving as the catalytic base to accept the proton delivered by the substrate. Our results also revealed that AceK prefers a single Mg2+-containing active site in the phosphotransfer reaction. The catalytic roles of conserved residues in the active site are discussed.

Crystal Structures of Carbamate Kinase from Giardia lamblia Bound with Citric Acid and AMP-PNP

The parasite Giardia lamblia utilizes the L-arginine dihydrolase pathway to generate ATP from L-arginine. Carbamate kinase (CK) catalyzes the last step in this pathway, converting ADP and carbamoyl phosphate to ATP and ammonium carbamate. Because the L-arginine pathway is essential for G. lamblia survival and absent in high eukaryotes including humans, the enzyme is a potential target for drug development. We have determined two crystal structures of G. lamblia CK (glCK) with bound ligands. One structure, in complex with a nonhydrolyzable ATP analog, adenosine 5′-adenylyl-β,γ-imidodiphosphate (AMP-PNP), was determined at 2.6 Å resolution. The second structure, in complex with citric acid bound in the postulated carbamoyl phosphate binding site, was determined in two slightly different states at 2.1 and 2.4 Å resolution. These structures reveal conformational flexibility of an auxiliary domain (amino acid residues 123–170), which exhibits open or closed conformations or structural disorder, depending on the bound ligand. The structures also reveal a smaller conformational change in a region associated the AMP-PNP adenine binding site. The protein residues involved in binding, together with a model of the transition state, suggest that catalysis follows an in-line, predominantly dissociative, phosphotransfer reaction mechanism, and that closure of the flexible auxiliary domain is required to protect the transition state from bulk solvent.

Calorimetric and spectroscopic investigation of the interaction between the C‐terminal domain of Enzyme I and its ligands

Enzyme I initiates a series of phosphotransfer reactions during sugar uptake in the bacterial phosphotransferase system. Here, we have isolated a stable recombinant C-terminal domain of Enzyme I (EIC) of Escherichia coli and characterized its interaction with the N-terminal domain of Enzyme I (EIN) and also with various ligands. EIC can phosphorylate EIN, but their binding is transient regardless of the presence of phosphoenolpyruvate (PEP). Circular dichroism and NMR indicate that ligand binding to EIC induces changes near aromatic groups but not in the secondary structure of EIC. Binding of PEP to EIC is an endothermic reaction with the equilibrium dissociation constant (K(D) ) of 0.28 mM, whereas binding of the inhibitor oxalate is an exothermic reaction with K(D) of 0.66 mM from calorimetry. The binding thermodynamics of EIC and PEP compared to that of Enzyme I (EI) and PEP reveals that domain-domain motion in EI can contribute as large as ∼-3.2 kcal/mol toward PEP binding.

Aerobic glycerol dissimilation via the Enterococcus faecalis DhaK pathway depends on NADH oxidase and a phosphotransfer reaction from PEP to DhaK via EIIADha

Two pathways for glycerol dissimilation are present in Enterococcus faecalis. Either glycerol is first phosphorylated by glycerol kinase and then oxidized by glycerol-3-phosphate oxidase with molecular oxygen as the electron acceptor (GlpO/GlpK pathway), or it is first oxidized by glycerol dehydrogenase with NAD(+) as the acceptor of the reduction equivalents and then phosphorylated by dihydroxyacetone kinase (GldA/DhaK pathway). The final end product in both cases is dihydroxyacetone phosphate (DHAP). The genes of the GldA/DhaK pathway are present in a four-gene operon structure encoding GldA, a small hypothetical protein (EF1359), and two subunits of dihydroxyacetone kinase (DhaK and DhaL). We demonstrate in this study that protein EF1359 is part of a phosphorylation cascade which phosphorylates dihydroxyacetone in a phosphoenolpyruvate (PEP)-dependent reaction via EI, HPr, EF1359 and DhaLK. Furthermore we show that aerobic dissimilation of glycerol via the GldA/DhaK pathway is dependent on active NADH oxidase to regenerate NADH in Ent. faecalis. A refined model of the aerobic metabolism of glycerol via the GldA/DhaK pathway is presented.

Solution Structure of the IIAChitobiose-HPr Complex of the N,N′-Diacetylchitobiose Branch of the Escherichia coli Phosphotransferase System

The solution structure of the complex of enzyme IIA of the N,N'-diacetylchitobiose (Chb) transporter with the histidine phosphocarrier protein HPr has been solved by NMR. The IIA(Chb)-HPr complex completes the structure elucidation of representative cytoplasmic complexes for all four sugar branches of the bacterial phosphoryl transfer system (PTS). The active site His-89 of IIA(Chb) was mutated to Glu to mimic the phosphorylated state. IIA(Chb)(H89E) and HPr form a weak complex with a K(D) of ~0.7 mM. The interacting binding surfaces, concave for IIA(Chb) and convex for HPr, complement each other in terms of shape, residue type, and charge distribution, with predominantly hydrophobic residues, interspersed by some uncharged polar residues, located centrally, and polar and charged residues at the periphery. The active site histidine of HPr, His-15, is buried within the active site cleft of IIA(Chb) formed at the interface of two adjacent subunits of the IIA(Chb) trimer, thereby coming into close proximity with the active site residue, H89E, of IIA(Chb). A His89-P-His-15 pentacoordinate phosphoryl transition state can readily be modeled without necessitating any significant conformational changes, thereby facilitating rapid phosphoryl transfer. Comparison of the IIA(Chb)-HPr complex with the IIA(Chb)-IIB(Chb) complex, as well as with other cytoplasmic complexes of the PTS, highlights a unifying mechanism for recognition of structurally diverse partners. This involves generating similar binding surfaces from entirely different underlying structural elements, large interaction surfaces coupled with extensive redundancy, and side chain conformational plasticity to optimize diverse sets of intermolecular interactions.

The Nla28S/Nla28 Two-Component Signal Transduction System Regulates Sporulation in Myxococcus xanthus

The response regulator Nla28 is a key component in a cascade of transcriptional activators that modulates expression of many important developmental genes in Myxococcus xanthus. In this study, we identified and characterized Nla28S, a histidine kinase that modulates the activity of this important regulator of M. xanthus developmental genes. We show that the putative cytoplasmic domain of Nla28S has the in vitro biochemical properties of a histidine kinase protein: it hydrolyzes ATP and undergoes an ATP-dependent autophosphorylation that is acid labile and base stable. We also show that the putative cytoplasmic domain of Nla28S transfers a phosphoryl group to Nla28 in vitro, that the phosphotransfer is specific, and that a substitution in the predicted site of Nla28 phosphorylation (aspartate 53) abolishes the phosphotransfer reaction. In phenotypic studies, we found that a mutation in nla28S produces a developmental phenotype similar to, but weaker than, that produced by a mutation in nla28; both mutations primarily affect sporulation. Together, these data indicate that Nla28S is the in vivo histidine kinase partner of Nla28 and that the primary function of the Nla28S/Nla28 two-component signal transduction system is to regulate sporulation genes. The results of genetic studies suggest that phosphorylation of Nla28S is important for the in vivo sporulation function of the Nla28S/Nla28 two-component system. In addition, the quorum signal known as A-signal is important for full developmental expression of the nla28S-nla28 operon, suggesting that quorum signaling regulates the availability of the Nla28S/Nla28 signal transduction circuit in developing cells.

A new variant of phosphoglycerate kinase deficiency (p.I371K) with multiple tissue involvement: Molecular and functional characterization

Phosphoglycerate kinase (PGK) is a key glycolytic enzyme that catalyzes the reversible phosphotransfer reaction from 1,3-bisphosphoglycerate to MgADP, to form 3-phosphoglycerate and MgATP. Two isozymes encoded by distinct genes are present in humans: PGK-1, located on Xq-13.3, encodes a ubiquitous protein of 417 amino acids, whereas PGK-2 is testis-specific.PGK1 deficiency is characterized by mild to severe hemolytic anemia, neurological dysfunctions and myopathy; patients rarely exhibit all three clinical features. Nearly 40 cases have been reported, 27 of them characterized at DNA or protein level, and 20 different mutations were described.Here we report the first Italian case of PGK deficiency characterized at a molecular and biochemical level. The patient presented during infancy with hemolytic anemia, increased CPK values, and respiratory distress; the study of red blood cell enzymes showed a drastic reduction in PGK activity. In adulthood he displayed mild hemolytic anemia, mental retardation and severe myopathy.PGK-1 gene sequencing revealed the new missense mutation c.1112T>A (p.Ile371Lys). The mutation was not found among 100 normal alleles, and even if located in the third to the last nucleotide of exon 9, it did not alter mRNA splicing. The p.Ile371Lys mutation falls in a conserved region of the enzyme, near the nucleotide binding site. The mutant enzyme shows reduced catalytic rates toward both substrates (apparent kcat values, 12-fold lower than wild-type) and a decreased affinity toward MgATP (apparent Km, 6-fold higher than wild-type). Moreover, it lost half of activity after nearly 9-min incubation at 45°C, a temperature that did not affect the wild-type enzyme (t1/2>1h). The possible compensatory expression of PGK2 isoenzyme was investigated in the proband and in the heterozygote healthy sisters, and found to be absent. Therefore, the highly perturbed catalytic properties of the new variant p.Ile371Lys, combined with protein instability, account for the PGK deficiency found in the patient and correlate with the clinical expression of the disease.

Metabolism of circulating ADP in the bloodstream is mediated via integrated actions of soluble adenylate kinase‐1 and NTPDase1/CD39 activities

Extracellular ATP and ADP trigger inflammatory, vasodilatatory, and prothrombotic signaling events in the vasculature, and their turnover is governed by networks of membrane-associated enzymes. The contribution of soluble activities to intravascular nucleotide homeostasis remains controversial. By using thin-layer chromatographic assays, we revealed transphosphorylation of [γ-(32)P]ATP and AMP by human and murine sera, which was progressively inhibited by specific adenylate kinase (AK) inhibitor Ap(5)A. This phosphotransfer reaction was diminished markedly in serum from knockout mice lacking the major AK isoform, AK1, and in human serum immunodepleted of AK1. We also showed that ∼75% ADP in cell-free serum is metabolized via reversible AK1 reaction 2ADP ↔ ATP + AMP. The generated ATP and AMP are then metabolized through the coupled nucleotide pyrophosphatase/phosphodiesterase and 5'-nucleotidase/CD73 reactions, respectively. Constitutive presence of another nucleotide-converting enzyme, nucleoside triphosphate diphosphohydrolase-1 (NTPDase1, known as CD39), was ascertained by the relative deficiency of serum from CD39-null mice to dephosphorylate [(3)H]ADP and [γ-(32)P]ATP, and also by diminished [(3)H]ADP hydrolysis by human serum pretreated with NTPDase1 inhibitors, POM-1 and ARL-67156. In summary, we have identified hitherto unrecognized soluble forms of AK1 and NTPDase1/CD39 that contribute in the active cycling between the principal platelet-recruiting agent ADP and other circulating nucleotides.

The plastidial glucan, water dikinase (GWD) catalyses multiple phosphotransfer reactions

The plant genome encodes at least two distinct and evolutionary conserved plastidial starch-related dikinases that phosphorylate a low percentage of glucosyl residues at the starch granule surface. Esterification of starch favours the transition of highly ordered α-glucans to a less ordered state and thereby facilitates the cleavage of interglucose bonds by hydrolases. Metabolically most important is the phosphorylation at position C6, which is catalysed by the glucan, water dikinase (GWD). The reactions mediated by recombinant wild-type GWD from Arabidopsis thaliana (AtGWD) and from Solanum tuberosum (StGWD) were studied. Two mutated proteins lacking the conserved histidine residue that is indispensible for glucan phosphorylation were also included. The wild-type GWDs consume approximately 20% more ATP than is required for glucan phosphorylation. Similarly, although incapable of phosphorylating α-glucans, the two mutated dikinase proteins are capable of degrading ATP. Thus, consumption of ATP and phosphorylation of α-glucans are not strictly coupled processes but, to some extent, occur as independent phosphotransfer reactions. As revealed by incubation of the GWDs with [γ-(33) P]ATP, the consumption of ATP includes the transfer of the γ-phosphate group to the GWD protein but this autophosphorylation does not require the conserved histidine residue. Thus, the GWD proteins possess two vicinal phosphorylation sites, both of which are transiently phosphorylated. Following autophosphorylation at both sites, native dikinases flexibly use various terminal phosphate acceptors, such as water, α-glucans, AMP and ADP. A model is presented describing the complex phosphotransfer reactions of GWDs as affected by the availability of the various acceptors.

Active site phosphoryl groups in the biphosphorylated phosphotransferase complex reveal dynamics in a millisecond time scale

The N-terminal domain of Enzyme I (EIN) and phosphocarrier HPr can form a biphosphorylated complex when they are both phosphorylated by excess cellular phosphoenolpyruvate. Here we show that the electrostatic repulsion between the phosphoryl groups in the biphosphorylated complex results in characteristic dynamics at the active site in a millisecond time scale. The dynamics is localized to phospho-His15 and the stabilizing backbone amide groups of HPr, and does not impact on the phospho-His189 of EIN. The dynamics occurs with the kex of ∼500s−1 which compares to the phosphoryl transfer rate of ∼850s−1 between EIN and HPr. The conformational dynamics in HPr may be important for its phosphotransfer reactions with multiple partner proteins. Structured summary of protein interactionsEIN and HPr bind by nuclear magnetic resonance (View Interaction).

Abstract 4171: Functional relevance of particular residues within the BRAF kinase domain in anaplastic thyroid carcinoma

Abstract The BRAFV600E mutation represents the most common oncogenic event in sporadic papillary thyroid cancer (PTC). Although at a much lower frequency, different missense point mutations (K601E, G474R, G469R) and complex genetic alterations, such as rearrangements (AKAP9-BRAF) or frameshift mutations (V600EK601del, V599ins, V600D-FLAGT601-605ins, T599I-VKSRdel), have also been reported in PTC. In contrast with PTC, no other alteration different from the V600E substitution has been shown in anaplastic thyroid carcinomas (ATC). In this study, we sought to determine the functional relevance of particular aminoacid residues, within the BRAF kinase domain, that we found mutated in some ATC. Fifty-four ATC were investigated for the existence of mutations at the activation loop, the glicine-rich phosphate-binding loop and the AKT binding sites of BRAF by means of PCR-SSCP and direct sequence. The structural-conformational changes behind punctual, unreported BRAF mutations and their effect on BRAF catalytic activity, substrate binding and downstream MAPK signalling were evaluated. Besides the V600E mutation, two additional point mutations (G615E and G474R) were detected within the BRAF kinase domain. The prevalence of both mutations, G615E (11% = 6 ATC) and G474R (7.4% = 4 ATC), was much lower than that of V600E (20% = 11 ATC). Interestingly, structural biology studies showed that G615E and G474R induced profound conformational changes within the BRAF kinase domain that resulted in reduced catalytic activity, decreased substrate binding and impaired downstream signalling. The G474R mutant, by preventing the N lobe from folding into a well ordered beta sheet structure, caused a broken or distorted phosphorylation-loop (P-loop). Because the P-loop is pivotal for ATP coordination and phosphotransfer reactions, the kinase activity of the BRAF G474R mutant was severely compromised and BRAF was unable to phosphorylate the downstream substrate. The G615E mutant by disrupting the structural integrity of the activation segment in the C lobe could in addition inhibit substrate binding. Significantly, none of the ATC bearing the G615E or the G474R mutation expressed the phospho-p44/42 MAPK (Thr202/Tyr204), which specifically recognizes the dually phosphorylated and active forms of ERK1 and ERK2. This observation not only supported the existence of impaired BRAF kinase activity in vivo but also suggested the abrogation of intracellular MEK/ERK signalling in those tumours. In summary, this study demonstrates that G615E and G474R behave similarly to D594V and the kinase-dead BRAFK483M mutant, knocking-down BRAF catalytic activity and downstream MEK/ERK signalling. Clinical trials involving the inhibitor of threonine/serine kinases Sorafenib or kinase specific inhibitors targeting constitutively active BRAF will prove inefficient therapeutic strategies in patients with G615E or G474R mutations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4171. doi:1538-7445.AM2012-4171

Protein-protein interactions between histidine kinases and response regulators of Mycobacterium tuberculosis H37Rv

Using yeast two-hybrid assay, we investigated protein-protein interactions between all orthologous histidine kinase (HK)/response regulator (RR) pairs of M. tuberculosis H37Rv and identified potential protein-protein interactions between a noncognate HK/RR pair, DosT/NarL. The protein interaction between DosT and NarL was verified by phosphotransfer reaction from DosT to NarL. Furthermore, we found that the DosT and DosS HKs, which share considerable sequence similarities to each other and form a two-component system with the DosR RR, have different cross-interaction capabilities with NarL: DosT interacted with NarL, while DosS did not. The dimerization domains of DosT and DosS were shown to be sufficient to confer specificity for DosR, and the different cross-interaction abilities of DosS and DosT with NarL were demonstrated to be attributable to variations in the amino acid sequences of the α2-helices of their dimerization domains.

Rotate into shape: MreB and bacterial morphogenesis

Rotate into shape: MreB and bacterial morphogenesis

Molecular characterization of the First Italian Variant of Phosphoglycerate Kinase Deficiency

Abstract 5270Phosphoglycerate kinase (PGK) is a key glycolytic enzyme that catalyzes the reversible phosphotransfer reaction from 1,3-bisphosphoglycerate (1,3-BPG) to ADP to form 3-phosphoglycerate (3-PG) and ATP. It is a small monomeric molecule characterized by two hinge-bent domains, with a highly conserved structure. The N-terminal domain binds 1,3-BPG or 3-PG, whereas the C-terminal domain binds Mg-ADP or Mg-ATP. Two isozymes, PGK1 and PGK2, are present in humans, encoded by two distinct genes. Whereas PGK2 is a testis-specific enzyme, PGK1 is expressed in all the somatic cells.The PGK1 gene is located on the Xq-13.1 chromosome, and encodes a protein of 416 amino acids. Mutations of the PGK1 gene result in enzyme deficiency characterized at clinical level by mild to severe hemolytic anemia, neurological dysfunctions and myopathy. Patients rarely exhibit all three clinical features. Since the first description by Kraus et al, nearly 40 patients with PGK deficiency have been reported, 27 of them characterized at the DNA or protein level. To date, 20 different mutations with worldwide distribution have been described.The aim of the study was to characterize the molecular defect in an Italian patient affected by phosphoglycerate kinase deficiency.The patient, born from unrelated parents with negative family history of neurological defects, showed at birth neonatal jaundice. At the age of four years, in concomitance of an infective episode, he displayed hemolytic anemia (Hb 8.6 g/dL, reticulocytes 19%, unconjugated bilirubin 0.91 mg/dL, LDH 445 u/l, aptoglobin absent) and increased CPK values (2483U/L). The patient showed respiratory distress. The study of red cell glycolytic enzymes displayed a drastic reduction of PGK activity (41.8 UI/gHb ref. values 287–392). We examined again the patient at the age of 25 yrs in occasion of his sister’s first pregnancy. The patient displayed compensated hemolytic anemia (Hb 14.1 g/dL, reticulocytes 6.6%) and severe myopathy.Sequencing analysis of the entire coding region and flanking intronic sequences of PGK1 gene showed the presence of a novel missense mutation c.1112 (ATA>AAA) responsible for amino acid substitution Ile371Lys. Although the mutation falls in the third last nucleotide of exon 9, it doesn’t alter the splicing as confirmed by patient cDNA analysis that showed a normal transcript. The new mutation is located in a highly conserved region among species close to the ATP binding site and it was not found among the 100 normal alleles examined thus excluding the possibility of a polymorphism. Family study performed in the parents, the two healthy sisters and maternal uncle confirmed the X-linked transmission of the disease. Disclosures:No relevant conflicts of interest to declare.

Identification of Critical Residues of the Mycobacterial Dephosphocoenzyme A Kinase by Site-Directed Mutagenesis

Dephosphocoenzyme A kinase performs the transfer of the γ-phosphate of ATP to dephosphocoenzyme A, catalyzing the last step of coenzyme A biosynthesis. This enzyme belongs to the P-loop-containing NTP hydrolase superfamily, all members of which posses a three domain topology consisting of a CoA domain that binds the acceptor substrate, the nucleotide binding domain and the lid domain. Differences in the enzymatic organization and regulation between the human and mycobacterial counterparts, have pointed out the tubercular CoaE as a high confidence drug target (HAMAP database). Unfortunately the absence of a three-dimensional crystal structure of the enzyme, either alone or complexed with either of its substrates/regulators, leaves both the reaction mechanism unidentified and the chief players involved in substrate binding, stabilization and catalysis unknown. Based on homology modeling and sequence analysis, we chose residues in the three functional domains of the enzyme to assess their contributions to ligand binding and catalysis using site-directed mutagenesis. Systematically mutating the residues from the P-loop and the nucleotide-binding site identified Lys14 and Arg140 in ATP binding and the stabilization of the phosphoryl intermediate during the phosphotransfer reaction. Mutagenesis of Asp32 and Arg140 showed catalytic efficiencies less than 5–10% of the wild type, indicating the pivotal roles played by these residues in catalysis. Non-conservative substitution of the Leu114 residue identifies this leucine as the critical residue from the hydrophobic cleft involved in leading substrate, DCoA binding. We show that the mycobacterial enzyme requires the Mg2+ for its catalytic activity. The binding energetics of the interactions of the mutant enzymes with the substrates were characterized in terms of their enthalpic and entropic contributions by ITC, providing a complete picture of the effects of the mutations on activity. The properties of mutants defective in substrate recognition were consistent with the ordered sequential mechanism of substrate addition for CoaE.