scholarly journals The Importance of Polarity in the Evolution of the K+ Binding Site of Pyruvate Kinase

2014 ◽  
Vol 15 (12) ◽  
pp. 22214-22226 ◽  
Author(s):  
Leticia Ramírez-Silva ◽  
Carlos Guerrero-Mendiola ◽  
Nallely Cabrera
Keyword(s):  
Binding Site ◽  
Pyruvate Kinase

scholarly journals Evolutionary plasticity in the allosteric regulator-binding site of pyruvate kinase isoform PykA from Pseudomonas aeruginosa

2019 ◽  
Vol 294 (42) ◽  
pp. 15505-15516 ◽  
Author(s):  
Yassmin Abdelhamid ◽  
Paul Brear ◽  
Jack Greenhalgh ◽  
Xavier Chee ◽  
Taufiq Rahman ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Binding Site ◽  
Pyruvate Kinase ◽  
Allosteric Regulator

Six Previously Undescribed Pyruvate Kinase Mutations Causing Enzyme Deficiency

Blood ◽  
1998 ◽  
Vol 92 (2) ◽  
pp. 647-652
Author(s):  
Anna Demina ◽  
Kottayil I. Varughese ◽  
José Barbot ◽  
Linda Forman ◽  
Ernest Beutler
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
Pyruvate Kinase ◽  
Hemolytic Anemia ◽  
Enzyme Deficiency ◽  
Sufficient Data ◽  
Common Cause ◽  
Pyruvate Kinase Deficiency ◽  
Substrate Binding Site ◽  
Local Perturbations

Erythrocyte pyruvate kinase deficiency is the most common cause of hereditary nonspherocytic hemolytic anemia. We present 6 previously undescribed mutations of the PKLR gene associated with enzyme deficiency located at cDNA nt 476 G→T (159Gly→Val), 884 C→T (295Ala→Val), 943 G→A (315Glu→Lys), 1022 G→A (341Gly→Asp), 1511 G→T (504Arg→Leu), and 1528 C→T (510Arg→Ter). Two of these mutations are near the substrate binding site: the 315Glu→Lys (943A) mutation may be involved in Mg2+ binding and159Gly→Val (476T) mutation has a possible effect on ADP binding. Four of six mutations produce deduced changes in the shape of the molecule. Two of these mutations,504Arg→Leu (1511T) and510Arg→Ter (1528T), are located at the interface of domains A and C. One of them (510Arg→Ter) is a deletion of the C-terminal residues affecting the integrity of the protein. The 504Arg→Leu mutation eliminates a stabilizing interaction between domains A and C. Changes in amino acid 341(nt 1022) from Gly to Asp cause local perturbations. The mutation295Ala→Val (884T) might affect the way pyruvate kinase interacts with other molecules. We review previously described mutations and conclude that there is not yet sufficient data to allow us to draw conclusions regarding genotype/phenotype relationship.

scholarly journals Analysis by cell-free transcription of the liver-specific pyruvate kinase gene promoter.

1989 ◽  
Vol 9 (10) ◽  
pp. 4409-4415 ◽  
Author(s):  
S Vaulont ◽  
N Puzenat ◽  
A Kahn ◽  
M Raymondjean
Keyword(s):  
Binding Site ◽  
Pyruvate Kinase ◽  
Gene Promoter ◽  
Specific Factor ◽  
Nuclear Factor ◽  
Flanking Sequence ◽  
Kinase Gene ◽  
Cell Extracts ◽  
Nuclear Factor 1

A DNA fragment spanning nucleotides -183 to -4 with respect to the cap site of the rat L-type pyruvate kinase (L-PK) gene contains at least four binding sites for putative transcriptional factors: hepatocyte nuclear factor 1 (HNF1), liver factor A1 (LF-A1), nuclear factor 1 (NF1), and major late transcription factor (MLTF). This fragment was used to direct transcription of a reporter sequence (a G-free cassette) in cell extracts. This L-PK promoter was active in liver nuclear extracts, but not in extracts from nonhepatic tissues. A reduction of 50% of the activity was obtained with a deleted L-PK promoter containing only the HNF1-binding site. In contrast, deletion of the HNF1-binding site inactivated the promoter by more than 90%. These results were confirmed by titration experiments with synthetic oligonucleotides. Titration of HNF1 resulted in an 85% decrease of transcriptional activity, while titration of LF-A1 resulted in only a 40% decrease. The influence of NF1 and MLTF seemed to be marginal in this system. The proximal 5'-flanking sequence of the L-PK gene therefore appears to function in vitro as an efficient liver-specific promoter which requires the binding of the liver factor HNF1 and which is also stimulated by the binding of another liver-specific factor, LF-A1.

Analysis by cell-free transcription of the liver-specific pyruvate kinase gene promoter

1989 ◽  
Vol 9 (10) ◽  
pp. 4409-4415
Author(s):  
S Vaulont ◽  
N Puzenat ◽  
A Kahn ◽  
M Raymondjean
Keyword(s):  
Binding Site ◽  
Pyruvate Kinase ◽  
Gene Promoter ◽  
Specific Factor ◽  
Nuclear Factor ◽  
Flanking Sequence ◽  
Kinase Gene ◽  
Cell Extracts ◽  
Nuclear Factor 1

A DNA fragment spanning nucleotides -183 to -4 with respect to the cap site of the rat L-type pyruvate kinase (L-PK) gene contains at least four binding sites for putative transcriptional factors: hepatocyte nuclear factor 1 (HNF1), liver factor A1 (LF-A1), nuclear factor 1 (NF1), and major late transcription factor (MLTF). This fragment was used to direct transcription of a reporter sequence (a G-free cassette) in cell extracts. This L-PK promoter was active in liver nuclear extracts, but not in extracts from nonhepatic tissues. A reduction of 50% of the activity was obtained with a deleted L-PK promoter containing only the HNF1-binding site. In contrast, deletion of the HNF1-binding site inactivated the promoter by more than 90%. These results were confirmed by titration experiments with synthetic oligonucleotides. Titration of HNF1 resulted in an 85% decrease of transcriptional activity, while titration of LF-A1 resulted in only a 40% decrease. The influence of NF1 and MLTF seemed to be marginal in this system. The proximal 5'-flanking sequence of the L-PK gene therefore appears to function in vitro as an efficient liver-specific promoter which requires the binding of the liver factor HNF1 and which is also stimulated by the binding of another liver-specific factor, LF-A1.

Functional characterization of the L-type pyruvate kinase gene glucose response complex

1993 ◽  
Vol 13 (12) ◽  
pp. 7725-7733
Author(s):  
M J Diaz Guerra ◽  
M O Bergot ◽  
A Martinez ◽  
M H Cuif ◽  
A Kahn ◽  
...  
Keyword(s):  
Binding Site ◽  
Pyruvate Kinase ◽  
Binding Sites ◽  
Ex Vivo ◽  
Functional Characterization ◽  
Insulin Response ◽  
Kinase Gene ◽  
Glucose Response ◽  
E Boxes ◽  
Glucose Responsiveness

L-type pyruvate kinase (L-PK) gene expression is modulated by hormonal and nutritional conditions. We have previously shown that the glucose/insulin response element (GlRE) of the L-PK gene is built around two noncanonical E boxes (element L4) that cooperate closely with a contiguous binding site (element L3). We present in this report the identification of proteins that interact with both elements. The L3 site binds hepatocyte nuclear factor 4 (HNF4)- and COUP/TF-related proteins. In fibroblasts, the overexpression of HNF4 transactivates the L-PK promoter. On the contrary, COUP/TF strongly inhibits the active promoter in hepatocytes. The L4 site binds the major late transcription factor (MLTF) in vitro and ex vivo; mutations that suppress this binding activity also inactivated the GlRE function. Mutations transforming one or two noncanonical E boxes of element L4 into consensus MLTF/USF binding sites strongly increase the affinity for MLTF/USF and do not impair the glucose responsiveness. However, merely the ability to bind MLTF/USF does not seem to be sufficient to confer a GlRE activity: those elements in which one E box has been destroyed and the other has been transformed into a consensus MLTF/USF sequence bind MLTF/USF efficiently but do not confer a high glucose responsiveness on the L-PK gene promoter. Consequently, the full activity of the L-PK GlRE seems to require the cooperation between two putative MLTF/USF binding sites located in the vicinity of an HNF4 binding site.

scholarly journals Functional characterization of the L-type pyruvate kinase gene glucose response complex.

1993 ◽  
Vol 13 (12) ◽  
pp. 7725-7733 ◽  
Author(s):  
M J Diaz Guerra ◽  
M O Bergot ◽  
A Martinez ◽  
M H Cuif ◽  
A Kahn ◽  
...  
Keyword(s):  
Binding Site ◽  
Pyruvate Kinase ◽  
Binding Sites ◽  
Ex Vivo ◽  
Functional Characterization ◽  
Insulin Response ◽  
Kinase Gene ◽  
Glucose Response ◽  
E Boxes ◽  
Glucose Responsiveness

L-type pyruvate kinase (L-PK) gene expression is modulated by hormonal and nutritional conditions. We have previously shown that the glucose/insulin response element (GlRE) of the L-PK gene is built around two noncanonical E boxes (element L4) that cooperate closely with a contiguous binding site (element L3). We present in this report the identification of proteins that interact with both elements. The L3 site binds hepatocyte nuclear factor 4 (HNF4)- and COUP/TF-related proteins. In fibroblasts, the overexpression of HNF4 transactivates the L-PK promoter. On the contrary, COUP/TF strongly inhibits the active promoter in hepatocytes. The L4 site binds the major late transcription factor (MLTF) in vitro and ex vivo; mutations that suppress this binding activity also inactivated the GlRE function. Mutations transforming one or two noncanonical E boxes of element L4 into consensus MLTF/USF binding sites strongly increase the affinity for MLTF/USF and do not impair the glucose responsiveness. However, merely the ability to bind MLTF/USF does not seem to be sufficient to confer a GlRE activity: those elements in which one E box has been destroyed and the other has been transformed into a consensus MLTF/USF sequence bind MLTF/USF efficiently but do not confer a high glucose responsiveness on the L-PK gene promoter. Consequently, the full activity of the L-PK GlRE seems to require the cooperation between two putative MLTF/USF binding sites located in the vicinity of an HNF4 binding site.

Six Previously Undescribed Pyruvate Kinase Mutations Causing Enzyme Deficiency

Blood ◽  
1998 ◽  
Vol 92 (2) ◽  
pp. 647-652 ◽  
Author(s):  
Anna Demina ◽  
Kottayil I. Varughese ◽  
José Barbot ◽  
Linda Forman ◽  
Ernest Beutler
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
Pyruvate Kinase ◽  
Hemolytic Anemia ◽  
Enzyme Deficiency ◽  
Sufficient Data ◽  
Common Cause ◽  
Pyruvate Kinase Deficiency ◽  
Substrate Binding Site ◽  
Local Perturbations

Abstract Erythrocyte pyruvate kinase deficiency is the most common cause of hereditary nonspherocytic hemolytic anemia. We present 6 previously undescribed mutations of the PKLR gene associated with enzyme deficiency located at cDNA nt 476 G→T (159Gly→Val), 884 C→T (295Ala→Val), 943 G→A (315Glu→Lys), 1022 G→A (341Gly→Asp), 1511 G→T (504Arg→Leu), and 1528 C→T (510Arg→Ter). Two of these mutations are near the substrate binding site: the 315Glu→Lys (943A) mutation may be involved in Mg2+ binding and159Gly→Val (476T) mutation has a possible effect on ADP binding. Four of six mutations produce deduced changes in the shape of the molecule. Two of these mutations,504Arg→Leu (1511T) and510Arg→Ter (1528T), are located at the interface of domains A and C. One of them (510Arg→Ter) is a deletion of the C-terminal residues affecting the integrity of the protein. The 504Arg→Leu mutation eliminates a stabilizing interaction between domains A and C. Changes in amino acid 341(nt 1022) from Gly to Asp cause local perturbations. The mutation295Ala→Val (884T) might affect the way pyruvate kinase interacts with other molecules. We review previously described mutations and conclude that there is not yet sufficient data to allow us to draw conclusions regarding genotype/phenotype relationship.

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