Phosphoglycerate kinase (PGK) catalyzes an important ATP-generating step in glycolysis. PGK1 deficiency is an uncommon X-linked inherited disorder, generally characterized by various combinations of non-spherocytic hemolytic anemia, neurological dysfunctions, and myopathies. Patients rarely exhibit all three clinical features. To provide a molecular framework to the different pathological manifestations, all known mutations were reviewed and 16 mutant enzymes, obtained as recombinant forms, were functionally and structurally characterized. Most mutations heavily affect thermal stability and to a different extent catalytic efficiency, in line with the remarkably low PGK activity clinically observed in the patients. Mutations grossly impairing protein stability, but moderately affecting kinetic properties (p.I47N, p.L89P, p.C316R, p.S320N, and p.A354P) present the most homogeneous correlation with the clinical phenotype. Patients carrying these mutations display hemolytic anemia and neurological disorders, and,except for p.A354P variant, no myopaty. Variants highly perturbed in both catalytic efficiency (p.G158V, p.D164V, p.K191del, D285V, p.D315N, and p.T378P) and heat stability (all, but p.T378P) result to be mainly associated with myopathy alone. Finally, mutations faintly affecting molecular properties (p.R206P, p.E252A, p.I253T, p.V266M, and p.D268N) correlate with a wide spectrum of clinical symptoms. These are the first studies that correlate the clinical symptoms with the molecular properties of the mutant enzymes. All findings indicate that the different clinical manifestations associated with PGK1 deficiency chiefly depend on the distinctive type of perturbations caused by mutations in the PGK1 gene, highlighting the need for determination of the molecular properties of PGK variants to assist in prognosis and genetic counseling. However, the clinical symptoms can not be understood only on the bases of molecular properties of the mutant enzyme. Different (environmental, metabolic, genetic and/or epigenetic) intervening factors can contribute toward the expression of PGK deficient clinical phenotypes.

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Phosphoglycerate kinase (PGK) is a key glycolytic enzyme that catalyzes the reversible transfer of a phoshoryl-group from 1,3-bisphosphoglycerate (1,3-BPG) to ADP forming 3-phosphoglycerate (3-PG) and ATP. PGK is a typical two-domain hinge-bending enzyme, with a highly conserved structure. The N-terminal domain binds 1,3-BPG/3-PG, whereas the C-terminal domain binds Mg-ADP/Mg-ATP.Humans have two PGK isozymes, PGK1 and PGK2, where PGK1 is an ubiquitous enzyme that is expressed in all somatic cells and PGK2 is a testis-specific enzyme. The PGK1 gene is located on the X-chromosome q-13.1, contains 11 exons and encodes a protein of 416 amino acids. Mutations of the PGK1 gene result in an enzyme deficiency that is for the most clinically characterized by mild-to severe hemolytic anemia and various defects in the central nervous system. To date, 19 different mutations with worldwide distribution have been reported. No correlation between the residual PGK activity and the severity of the clinical manifestations have been documented so far. To analyze the mutations at protein level and possibly to correlate the genotype to clinical phenotype, we started with the molecular characterization of the wild-type PGK1 enzyme and three mutants (I47N, D164 and S320N) obtained from E.coli as recombinant proteins. The corresponding mutations, i.e., c.140T>A, c.491A>T and c.959G>A, have been identified in patients with PGK deficiency and affected by severe hemolytic anemia and progressive mental retardation. The cDNA encoding the PGK1 was prepared starting from a blood sample of a healthy donor, with normal PGK1 activity. Site-directed mutagenesis was used to introduce the desired mutations into the PGK1 cDNA. The wild type enzyme was expressed to its maximum level (about 80–100 mg of enzyme per liter of culture) after 5 hours of induction with 0.5 mM IPTG at 37 °C. For mutant enzymes the induction temperature was lowered to 25°C. All recombinant enzymes were purified to homogeneity after a single chromatographic step on DEAE Sepharose column. The wild-type enzyme was crystallized in both free form or complexed with 3-PG. The corresponding structures were solved to high resolution (1.8 and 1.6 A, respectively) and compared. Essentially, binding 3-PG caused a 6° rotation of the N-domain in respect to the C-domain. The recombinant enzyme exhibited kinetic properties similar to those of the authentic enzyme, displaying vs 3-PG and ATP alike specific activities (about 1000 U/mg) and alike Km values (about 1mM). I47N and S320N mutant enzymes showed kcat values 3-fold lower than the wild-type enzyme. The D164V was characterized by a Km value vs 3-PG 15 times higher than that of the other enzymes studied and a catalytic efficiency 70 times lower. Finally, all mutant enzymes turned out to be highly heat unstable with respect to the wildtype enzyme, losing half of their activity after approximately 10 minutes of incubation at 37 °C. At higher temperatures, the wild-type enzyme was protected from heat inactivation by Mg-ATP or 3-PG. On the contrary, no one mutant was protect by Mg-ATP and the D164V and S320N mutants were not even protected by 3-PG. Therefore, these preliminary studies indicate that all mutations target amino acid residues located in positions primarily important for preserving the protein stability during the red cell life span.

Pyrimidine 5′-nucleotidase (P5′N-1) is a dephosphorylating enzyme that catalyzes the hydrolysis of various pyrimidine nucleoside 5′-monophosphates, particularly UMP and CMP, to produce the corresponding nucleosides. In RBC the reaction is essential for the removal of the nucleotides mainly arising from ribosomal RNA degradation during final erythroid maturation. Hereditary P5′N-1 deficiency is the third most common enzymopathy causing hereditary non-spherocytic hemolytic anemia. The disorder is transmitted as an autosomal recessive trait and is usually characterized by mild-to-moderate hemolytic anemia and accumulation of pyrimidine nucleotides within the erythrocyte. The enzyme is strongly inactivated by heavy metals; thus P5′N-1 deficiency can be acquired as a result of lead poisoning. The P5′N-1 gene is localized on 7p15-p14 and the cDNA has been cloned and sequenced. 24 different mutations have been identified so far, most of them at the homozygous level. Recently, five pathological variants of P5′N-1 have been in-depth characterized, and the molecular bases of the P5′N-1 deficiency has been elucidated. To unravel the cause of the P5′N-1 deficiency found in patients with hemolytic anemia and homozygous for 3 newly identified missense mutations (c.187T>C, c.469G>C, c.740T>C; Balta et al, Blood ASH2006, 108:3743; Manco et al, Haematologica2006, 91:266–267), we have undertaken a functional analysis of the 3 mutant enzymatic forms. The C63R, G157R and I247T proteins were produced as recombinant forms, purified and biochemically characterized. All enzymes were altered, although to a different extent, either in their catalytic efficiency or in thermal stability, the G157R being the most impaired enzyme. Catalytic efficiency of all mutants turned expecially towards UMP (about 50 to 200 times), owing to the increased Km values (about 10–25 times higher). The kinetic behaviour vs CMP was partly affected, the catalytic activity being moderately reduced (Kcat lowered to 5–20%). The G157R protein was highly heat unstable, halving the activity in about 23 min at 37°C, whereas C63R and I247T mutants at the same temperature maintained fully activity for more than 2 hours. However, at higher temperature also C63R and I247T mutants resulted less stable than the wild-type enzyme losing the activity in few minutes (t1/2 at 46°C, about 5 min vs 2 hours of the wild-type enzyme). Therefore, although mutations targeted different regions of the P5′N-1 structure, unexpectedly they produced similar aberrant effects on the molecular properties of the enzyme. Gly157 is a conserved amino acid, located close to the substrate binding site. Very likely, position 157 cannot tolerate the large and charged arginine side-chain introduced by c.469G>C mutation. Thus, it is conceivable that the drastic G157R substitution not only indirectly affects the binding of the substrate(s), but also weakens the protein stability. Cys63 and Ile247 are located far away from the catalytic site. Nevertheless, our biochemical data indicate that they are functionally and structurally important for preserving the enzyme activity. Thus, as in other cases, the decreased catalytic efficiency of C63R and I247T enzymes seems to result from secondary effects related to propagating conformational changes.

3016 Poster Board II-992 Phosphoglycerate 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 relatively 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. During the catalytic cycle, the enzyme undergoes large conformational rearrangements, proceeding from an open form to a closed form. 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 X-chromosome q-13.1, and encodes a protein of 416 amino acids. Mutations of the PGK1 gene result in an enzyme deficiency, that is characterized by mild to severe hemolytic anemia, neurological dysfunctions and myopathy. Patients rarely exhibit all three clinical features. To date, 20 different mutations with worldwide distribution have been described. To investigate the genotype-phenotype relationship of PGK deficiency, recently we have undertaken a characterization of the all PGK mutant enzymes so far reported. In this study we describe the molecular abnormalities of the G158V, R206P, V266M and D285V variants obtained from E.coli as recombinant proteins. All patients were affected by moderate to severe hemolytic anemia. Moreover, patients bearing GI58V, R206P, and D285V variants displayed muscular disorders. Neurological dysfunctions were present in patients with R206P and V266M. The desired mutations were introduced into the PGK cDNA by site directed mutagenesis. All mutant enzymes were expressed and purified to homogeneity as previously indicated (Morera et al., Blood, ASH, Annual Meeting Abstracts, 2008;112:2875). Each variant was subjected to kinetic analysis and to different heat treatments in the absence and in the presence of specific ligands. The enzyme activity was determined following the backward reaction. Variants G158V and D285V turned out to be affected in their catalytic activities, displaying kcat values towards ATP and 3-PG 7-fold and 19-fold, respectively, lower than that of the wild type enzyme previously characterized. Variant R206P displayed reduced affinity vs 3-PG, the Km value being 8-fold higher than that of the wild type. Variant V266M showed kinetic properties similar to those of the wild type. The mutant enzymes subjected to heat treatments exhibited different protein stability. Whereas the wild type enzyme preserved 70% of its activity after one hour-incubation at 45°C, mutants G158V and D285V at the same temperature halved their activities after only 5 min and 2 min, respectively. Mutants R206P and V266M turned to be quite heat stable, their T50 (the temperature to which an enzyme halves its activity in 10 min) being 2°C lower than that of the wild type enzyme (47°C vs 49°C). Moreover, at a temperature 3-4 °C higher than its own T50, no one mutant was properly protected by the presence of Mg-ATP. In addition, variants G158V and D285V were not even protected by 3-PG. Therefore, these studies suggest that G158V and D285V substitutions affect amino acid residues located in key positions for allowing the enzyme to preserve its protein stability, especially during the red cell life span, and to adopt its proper conformations in fulfilling the catalytic cycle. The reduced RBC concentration of PGK and the energy pathway deficiency would account for the dysfunctions displayed by patients with G158V and D285V. With regard to R206P variant, the mutation affects an amino acid residue located in the hinge of the enzyme, far away from the 3-PG binding site. Owing to the fact that the variant displayed a reduced affinity versus 3-PG, it is likely that Arg206 plays an important role in the structuring of the 3-PG binding site, via long-distance interactions. Thus mutation R206P would lead to a distortion of the 3-PG binding site, impairing the PGK activity under physiological 3-PG concentrations. Consequently, the reduced energy supply would be the cause of the hemolysis displayed by the PGK deficient patient. Finally, with regard to V266M mutant, no acceptable explanation of the enzyme deficiency can be drawn by the present biochemical studies, the mutant behaving as the wild type enzyme. Disclosures: No relevant conflicts of interest to declare.