scholarly journals Incomplete refolding of a fragment of the N-terminal domain of pig muscle 3-phosphoglycerate kinase that lacks a subdomain

2001 ◽  
Vol 268 (6) ◽  
pp. 1851-1860 ◽  
Author(s):  
Andrea N. Szilágyi ◽  
Nina V. Kotova ◽  
Gennady V. Semisotnov ◽  
Mária Vas
Keyword(s):  
Phosphoglycerate Kinase ◽  
Terminal Domain ◽  
Pig Muscle

scholarly journals Identification of new mutations in two phosphoglycerate kinase (PGK) variants expressing different clinical syndromes: PGK Creteil and PGK Amiens

Blood ◽  
1994 ◽  
Vol 84 (3) ◽  
pp. 898-903
Author(s):  
M Cohen-Solal ◽  
C Valentin ◽  
F Plassa ◽  
G Guillemin ◽  
F Danze ◽  
...  
Keyword(s):  
Mental Retardation ◽  
Amino Acid ◽  
Hemolytic Anemia ◽  
Amino Acid Substitution ◽  
Polymerase Chain Reaction Amplification ◽  
Phosphoglycerate Kinase ◽  
Terminal Domain ◽  
Clinical Consequences ◽  
Reaction Amplification ◽  
Chronic Hemolytic Anemia

Phosphoglycerate kinase (PGK) deficiency is generally associated with chronic hemolytic anemia, although it can be accompanied by either mental retardation or muscular disease. Genomic DNAs of two PGK- deficient patients previously described in France were sequenced directly after polymerase chain reaction amplification. The PGK Creteil variant arises from a G-->A nucleotide interchange at position 1022 in cDNA (exon 9), resulting in amino acid substitution 314 Asp-->Asn in the C-terminal domain, which contains the nucleotide binding site. It is associated with rhabdomyolysis crises but not with hemolysis or mental retardation. In the other case, which is associated with chronic hemolytic anemia and mental retardation (PGK Amiens), an A-->T nucleotide interchange was found at position 571 in cDNA (exon 5); this leads to amino acid substitution 163 Asp-->Val in the N-terminal domain, which contains the catalytic site for phosphoglycerate binding. These results corroborate the kinetic data observed. In the two cases, the mutations are distinct from others previously reported and no significant relationship could be observed between the location of the amino acid substitution and its clinical consequences.

Immobilization of pig muscle 3-phosphoglycerate kinase

1985 ◽  
Vol 7 (7) ◽  
pp. 357-360 ◽  
Author(s):  
L.M. Simon ◽  
J. Szelei ◽  
B. Szajáni ◽  
L. Boross
Keyword(s):  
Phosphoglycerate Kinase ◽  
Pig Muscle

Phosphoglycerate Kinase Deficiency: Characterization of the Wild-Type Enzyme and Three Pathological Variants Generated from C.140T>a, C.491A>T and C.959G>a Mutations

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2875-2875
Author(s):  
Simone Morera ◽  
Laurent Chiarelli ◽  
Stefano Rovida ◽  
Paola Bianchi ◽  
Elisa Fermo ◽  
...  
Keyword(s):  
Hemolytic Anemia ◽  
Clinical Manifestations ◽  
Phosphoglycerate Kinase ◽  
Free Form ◽  
Heat Inactivation ◽  
Kinetic Properties ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Terminal Domain

Abstract 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.

scholarly journals Protease digestion studies of an equilibrium intermediate in the unfolding of creatine kinase

1997 ◽  
Vol 321 (1) ◽  
pp. 83-88 ◽  
Author(s):  
Timothy WEBB ◽  
Philip. J. JACKSON ◽  
Glenn E. MORRIS
Keyword(s):  
Creatine Kinase ◽  
Guanidine Hydrochloride ◽  
Gel Filtration ◽  
Phosphoglycerate Kinase ◽  
Domain Model ◽  
Terminal Portion ◽  
Terminal Domain ◽  
Low Concentrations ◽  
Protease Digestion ◽  
Partial Unfolding

Protease digestion experiments have been used to characterize the structure of an equilibrium intermediate in the unfolding of creatine kinase (CK) by low concentrations (0.625 M) of guanidine hydrochloride (GdnHCl). Eighteen of the major products of digestion by trypsin, chymotrypsin and endoproteinase Glu-C have been identified by microsequencing after separation by SDS/PAGE and electroblotting on poly(vinylidene difluoride) membranes. The C-terminal portion (Gly215 to Lys380) was much more resistant to digestion than the N-terminal portion (Pro1 to Gly133), although the area most sensitive to proteolysis was in the middle of the CK sequence (Arg134 to Arg214). These experiments are consistent with the two-domain model for the CK monomer. The structure of the intermediate is proposed to consist of a folded C-terminal domain and a partly folded N-terminal domain separated by an unfolded central linker. Protease susceptibility is clustered within two N-terminal regions and one central region. These regions are evidently exposed as a result of the partial unfolding and/or separation of the N-terminal domain. Further evidence for the structure of this intermediate comes from gel filtration studies. Treatment of CK with 0.625 M GdnHCl resulted in slow aggregation at 37 °C, but not at 12 °C, a phenomenon previously reported for phosphoglycerate kinase. The aggregation did not occur at higher GdnHCl concentrations and was unaffected by a reducing agent. It is proposed that aggregation is a consequence of non-specific interactions between hydrophobic regions, possibly domain/domain interfaces, which become exposed in the intermediate.

Molecular characterization of the First Italian Variant of Phosphoglycerate Kinase Deficiency

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5270-5270
Author(s):  
Paola Bianchi ◽  
Elisa Fermo ◽  
Luana Mandarà ◽  
Cristina Vercellati ◽  
Anna Paola Maria Luisa Marcello ◽  
...  
Keyword(s):  
Hemolytic Anemia ◽  
Conflicts Of Interest ◽  
Phosphoglycerate Kinase ◽  
Glycolytic Enzyme ◽  
Molecular Defect ◽  
Sequencing Analysis ◽  
Coding Region ◽  
Atp Binding Site ◽  
Terminal Domain ◽  
Worldwide Distribution

Abstract Abstract 5270 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 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.

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