Human Muscle Phosphofructokinase Research Articles

Crystallization and preliminary crystallographic analysis of human muscle phosphofructokinase, the main regulator of glycolysis

Whereas the three-dimensional structure and the structural basis of the allosteric regulation of prokaryotic 6-phosphofructokinases (Pfks) have been studied in great detail, knowledge of the molecular basis of the allosteric behaviour of the far more complex mammalian Pfks is still very limited. The human muscle isozyme was expressed heterologously in yeast cells and purified using a five-step purification protocol. Protein crystals suitable for diffraction experiments were obtained by the vapour-diffusion method. The crystals belonged to space group P6222 and diffracted to 6.0 Å resolution. The 3.2 Å resolution structure of rabbit muscle Pfk (rmPfk) was placed into the asymmetric unit and optimized by rigid-body and group B-factor refinement. Interestingly, the tetrameric enzyme dissociated into a dimer, similar to the situation observed in the structure of rmPfk.

Electron microscopy analysis of mammalian phosphofructokinase reveals an unusual 3-dimensional structure with significant implications for enzyme function

Phosphofructokinase is a sophisticated allosteric enzyme that is fundamental for the control of glycolysis. The structure of the bacterial enzyme is well characterized. However, little is known about the structural organization of the more complex enzyme from mammals. We have obtained the structure of human muscle phosphofructokinase in the presence of fructose 6-phosphate at a resolution of 1.8 nm by electron microscopy (EM). Particles of the tetrameric enzyme corresponded to an elongated molecule (14.5 × 9 nm) arranged into 2 dimeric subdomains. Image analysis and 3-dimensional reconstruction showed the presence of a prominent channel in one of the dimers but not in the opposite one, revealing that they are in greatly different conformations. Fitting of bacterial structures into the EM model suggested disruption of the fructose 6-phosphate catalytic and the fructose 2,6-bisphophate allosteric sites in the cavity-containing dimer. Therefore, the reported structure might have major implications for the function of mammalian phosphofructokinase.

Subunit Interactions and Composition of the Fructose 6-Phosphate Catalytic Site and the Fructose 2,6-Bisphosphate Allosteric Site of Mammalian Phosphofructokinase

Mammalian phosphofructokinase originated by duplication, fusion, and divergence of a primitive prokaryotic gene, with the duplicated fructose 6-phosphate catalytic site in the C-terminal half becoming an allosteric site for the activator fructose 2,6-bisphosphate. It has been suggested that both sites are shared across the interface between subunits aligned in an antiparallel orientation, the N-terminal half of one subunit facing the C-terminal half of the other. The composition of these binding sites and the way in which subunits interact to form the dimer within the tetrameric enzyme have been reexamined by systematic point mutations to alanine of key amino acid residues of human muscle phosphofructokinase. We found that residues His-199, His-298, Arg-201, and Arg-292 contribute to the catalytic site and not to the allosteric site, because their mutation decreased the affinity for fructose 6-phosphate without affecting the activation by fructose 2,6-bisphosphate or its binding affinity. In contrast, residues Arg-566, Arg-655, and His-661 were critical components of the fructose bisphosphate allosteric site, because their mutation strongly reduced the action and affinity of the activator, with no alteration of substrate binding to the active site. Our results suggest that mammalian phosphofructokinase subunits associate with the N-terminal halves facing each other to form the two catalytic sites/dimer and the C-terminal halves forming the allosteric sites. Additionally, mutation of certain residues eliminated activation by fructose 1,6-bisphosphate, but not its binding, with little effect on activation by fructose 2,6-bisphosphate, indicating a divergence in the signal transduction route despite their binding to the same site.

The First Crystal Structure of Phosphofructokinase from a Eukaryote: Trypanosoma brucei

The crystal structure of the ATP-dependent phosphofructokinase (PFK) from Trypanosoma brucei provides the first detailed description of a eukaryotic PFK, and enables comparisons to be made with the crystal structures of bacterial ATP-dependent and PPi-dependent PFKs. The structure reveals that two insertions (the 17-20 and 329-348 loops) that are characteristic of trypanosomatid PFKs, but absent from bacterial and mammalian ATP-dependent PFKs, are located within and adjacent to the active site, and are in positions to play important roles in the enzyme's mechanism. The 90 residue N-terminal extension forms a novel domain that includes an “embracing arm” across the subunit boundary to the symmetry-related subunit in the tetrameric enzyme. Comparisons with the PPi-dependent PFK from Borrelia burgdorferi show that several features thought to be characteristic of PPi-dependent PFKs are present in the trypanosome ATP-dependent PFK. These two enzymes are generally more similar to each other than to the bacterial or mammalian ATP-dependent PFKs. However, there are critical differences at the active site of PPi-dependent PFKs that are sufficient to prevent the binding of ATP. This crystal structure of a eukaryotic PFK has enabled us to propose a detailed model of human muscle PFK that shows active site and other differences that offer opportunities for structure-based drug discovery for the treatment of sleeping sickness and other diseases caused by the trypanosomatid family of protozoan parasites.

Regulation of transcription from the human muscle phosphofructokinase P2 promoter by the Sp1 transcription factor.

The human muscle phosphofructokinase (HPFKM) p2 promoter contains sequence elements that are similar to the Sp1 transcription factor binding site consensus sequence. DNase I footprinting identified four regions of the HPFKM p2 promoter that bound purified Sp1. Gel retardation analysis using HeLa S3 nuclear extracts and purified Sp1 protein demonstrated that each of the four recognition elements bound the Sp1 transcription factor. The function of the HPFKM p2 promoter elements was examined in transient transfection assays using these binding sites cloned into a minimal promoter element. In Drosophila Schneider line-2 cells, each of these regulatory regions trans-activated transcription from a minimal promoter element in response to exogenously expressed Sp1. In addition, transcription from the HPFKM p2 promoter was shown to be trans-activated by exogenously expressed Sp1 in Drosophila Schneider line-2 cells. Deletion analysis of the HPFKM p2 promoter demonstrated that the promoter region between -66 and +16 was sufficient to confer sp1 responsiveness. This promoter region includes one of the regulatory elements footprinted by the purified Sp1 transcription factor and mediates the majority of the transcriptional activity from the HPFKM p2 promoter in the human cervical carcinoma cell line HeLa S3. This demonstrates that the HPFKM p2 promoter contains four functional Sp1 binding sites that may contribute to the level of transcription from this promoter in a variety of cell types.

Novel clustering of Sp1 transcription factor binding sites at the transcription initiation site of the human muscle phosphofructokinase P1 promoter.

The regulatory sequence elements of the human muscle phosphofructokinase (HPFKM) p1 promoter from -655 to +78 were cloned and characterized. In the human cervical carcinoma cell line, HeLa S3, the HPFKM type C RNA initiated from a single predominant transcription initiation site and the HPFKM p1 promoter displayed transcriptional activity in transient transfection assays. The HPFKM p1 promoter region was shown to possess eight binding sites for the Sp1 transcription factor by DNase I footprinting and gel retardation analysis. The functional importance of these interactions was examined by transient transfection analysis in Drosophila SL2 and HeLa S3 cells. This analysis demonstrated that the HPFKM p1 promoter sequence between +12 and +78 retained Sp1-dependent transcriptional activity in Drosophila SL2 cells and retained promoter activity in HeLa S3 cells. These results suggest that the Sp1 binding site (site 8 between +12 and +21) immediately adjacent to the transcription initiation site represents an important regulatory element of this promoter at least in the context of the minimal HPFKM p1 promoter. However mutagenesis of the Sp1 site 8 demonstrated that, in the context of a larger HPFKM p1 promoter region containing Sp1 sites 1 to 7, it now contributed very little to the total promoter activity. Therefore it appears the Sp1 sites in the HPFKM p1 promoter display functional redundancy.

Cloning and characterization of the human muscle phosphofructokinase gene.

A 35-kbp region of genomic DNA encoding the human muscle phosphofructokinase (HPFK-M) gene including all of the coding exons (1-22) plus 2.2-kbp of 5'-flanking sequence has been cloned. The exon boundaries are the same as has been observed for the rabbit muscle phosphofructokinase (RPFK-M), the human liver phosphofructokinase (HPFK-L), and the mouse liver phosphofructokinase (MPFK-L) genes. Characterization of the structure of the HPFK-M gene and its transcript in Epstein-Barr virus transformed B-cell lines derived from patients with glycogen storage disease type VII (GSDVII or Tarui's disease) demonstrated that this single-copy gene encodes a normal sized 3.0-kb transcript in the four cases examined. This suggests the lesion in these cases represents either a point mutation or possibly a small insertion or deletion resulting in the synthesis of a defective HPFK-M protein. Analysis of the 5'-flanking region demonstrated the presence of a functional promoter located within 114 nucleotides of a proposed transcription initiation site. This promoter was active in the human cervical carcinoma cell line, HeLa S3, the dedifferentiated human hepatoma cell line, HepG2.1, and the mouse myoblast cell line, C2C12, suggesting this promoter has a broad cell-type specificity. In addition, from the known HPFK-M cDNA sequences, this observation indicates that the HPFK-M gene has a second promoter located upstream from the genomic region isolated in this study.

Structure of the entire human muscle phosphofructokinase-encoding gene: a two-promoter system

We have recently shown that three types (A, B, and C) of mRNA species are transcribed from a single gene encoding human muscle phosphofructokinase(hPFK-M) through alternative splicing [Nakajima et al., Biochem. Biophys. Res. Commun. 166 (1990) 637–641]. To determine its complete structure and elucidate the mechanism of alternative RNA splicing, we isolated the hPFK-M gene, which spans about 30 kb, and contains 24 exons. Transcription start points were observed for both exon 1 and cxon 2 by S1 nuclcase protection assay and primer extension. Motifs of an Sp1-binding site were observed in the upstream region of exon 1 (promoter 1). A TATA-box-like sequence and a CAAT-box-like sequence were identified in the upstream region of exon 2 (promoter 2). Reporter assay revealed that the promoter 1 region was functional both in HeLa cells and myoblastic clonal cells, and that the promoter 2 region was active only in myoblastic cells. Motifs of M-CAT known as a muscle-specific enhancer, were observed in the promoter 2 region. These results indicated that the hPFK-M gene contains at least two promoter regions, facilitating the expression of the heterogeneous gene transcripts in a cell-type-specific manner.

Abnormal high-energy phosphate metabolism in human muscle phosphofructokinase deficiency

We studied the pattern of high-energy phosphate metabolism in five patients with phosphofructokinase deficiency (PFKD) and five healthy subjects (HS) during graded rhythmic handgrip performed for 5 min at 17, 33, 50, and 100% of maximal voluntary contraction (MVC). The range of MVC was similar in both groups. Force production was recorded, and intracellular concentrations of phosphorus compounds and pH were measured in the flexor digitorum profundus of the active forearm. At exercise intensities greater than or equal to 50% MVC, changes in concentrations of high-energy phosphate metabolites were abnormal in PFKD. During maximal effort, [ADP], calculated from the creatine kinase reaction, was 64.3 +/- 13.5 (SE) mumol/kg in PFKD vs. 25.7 +/- 4.0 in HS (P less than 0.05). Ammonia (NH3), a product of AMP deamination and an index of muscle [AMP], increased approximately twofold more in venous effluent during maximal forearm exercise in PFKD than in HS (P less than 0.05). Phosphocreatine concentration was 9.4 +/- 1.3 (SE) mmol/kg in HS and 13.0 +/- 1.7 in PFKD (P less than 0.05). Inorganic phosphate concentration was 15.8 +/- 1.4 mmol/kg in HS and 7.4 +/- 0.5 in PFKD (P less than 0.05). During strenuous exercise, PFKD patients exhibit an impairment in the rephosphorylation of ADP related to a subnormal oxidative capacity, an absence of glycolysis, and an attenuated breakdown of phosphocreatine.

Alternative splicing of the transcript encoding the human muscle isoenzyme of phosphofructokinase.

The genomic DNA encoding human muscle phosphofructokinase (HPFK-M) exons VII to X has been cloned and the coding regions have been sequenced. The intron/exon boundaries are located at the same positions as those identified for the rabbit phosphofructokinase-M gene (Lee, C. -P., Kao, M. -C., French, B. A., Putney, S. D., and Chang, S. H. (1987) J. Biol. Chem. 262, 4195-4199). A HPFK-M cDNA clone lacking the sequences corresponding to exon IX was isolated from a human fibroblast (IMR-90) library, suggesting that the HPFK-M transcript may be alternatively spliced. Exon IX is 93 nucleotides in length, and the absence of this sequence from the HPFK-M transcript would generate an RNA coding for a HPFK-M-related polypeptide lacking 31 amino acids compared with the HPFK-M polypeptide. HPFK-M transcripts approximately 3.0 kilobases in length are expressed in a tissue-specific manner with high levels in cell lines and skeltal muscle tissue and very low levels in peripheral blood mononuclear cells and liver tissue. Characterization of the structure of these HPFK-M transcripts by nuclease S1 and polymerase chain reaction analysis demonstrated that all the cell lines and tissues examined expressed the alternatively spliced transcript in addition to the transcript coding for the enzymatically functional HPFK-M polypeptide.

Evidence for alternative RNA splicing and possible alternative promoters in the human muscle phosphofructokinase gene at the 5′ untranslated region

Three mRNA species for human muscle phosphofructokinase containing heterogeneous 5′ untranslated sequences were identified through cDNA cloning. Type A mRNA was essentially the same as that reported previously ( Nakajima, H., et al. (1987) FEBS Lett. 223, 113). Type B mRNA was considered to be the major gene product, which contained an extra non-coding sequence within the 5′ untranslated region of type A mRNA. Amplification of mRNA by polymerase chain reaction revealed that types A and B mRNAs shared a common precursor RNA, and were alternatively spliced. Type C mRNA, homologous to the cDNA sequence from a placenta library ( Sharma, P. M. et al., (1989) Gene 77, 177), was considered to be under the control of an alternative promotor.

Cloning and expression of a human muscle phosphofructokinase cDNA

The nucleotide sequence of a 2.86-kb cDNA clone containing the complete human muscle phosphofructo-kinase (PFK) protein-coding region was determined. It comprises 76 bp of 5'-untranslated sequence, 2340 bp encoding human muscle PFK polypeptide, and 399 bp of 3'-untranslated sequence plus a poly(A) tract. A retroviral vector was utilized to express the product of this coding sequence in mouse fibroblasts. The PFK-coding cDNA was shown to code for an enzymatically active polypeptide by immunoprecipitation analysis and DEAE-Sephadex A-25 chromatography.

93 MOLECULAR ASPECT OF MYOGENIC HYPERURICEMIA: CLONING OF HUMAN MUSCLE PHOSPHOFRUCTOKINASE cDNA

Our recent investigations revealed that myogenic hyperuricemia (Mineo et al.) is a common pathologic feature of glycogenosis types III, V and VII, in which muscle glycolysis is impaired. To clarify the molecular defect in glycogenosis type VII (human muscle phosphofructokinase (HMPFK) deficiency), we began performing the molecular cloning of HMPFK cDNA. A human muscle cDNA library was prepared by Okayama and Berg method and screened by colony hybridization with a rabbit muscle PFK cDNA (a gift from Dr. S. D. Putney of the Repligen Corp. MA, U.S.A.). A partial HMPFK cDNA clone was isolated. The clone was primer-extended and two other overlapping clones to cover the full-length were isolated. Complete primary structure of the enzyme was determined through the sequence analysis. HMPFK was 85,050 Da in size with 779 amino acid residues. Internal homology between N- and C- halves of the peptide was observed as in rabbit muscle PFK. Sequence homologies between rabbit and human muscle PFK were 96% in amino acids and 89% in nucleotides. Cloning of HMPFK cDNA will help analyzing the pathological regulatory mechanism of muscle glycolysis responsible for the development of myogenic hyperuricemia at the molecular level.

Cloning of human muscle phosphofructokinase cDNA

Three overlapping cDNA clones for human muscle phosphofructokinase (HMPFK) covering the complete coding sequence were isolated. The sequence included a poly(A) tail, a 399 bp 3′-untranslated region, a 2337 bp coding region for 779 amino acid residues and a part of the 5′-untranslated region. Homologies between HMPFK and rabbit muscle phosphofructokinase (RMPFK) were 96% of the amino acids and 89% of the nucleotides in the coding region. Like RMPFK, HMPFK also possessed the internal homology between C- and N-halves in its primary structure. Cloning of HMPFK cDNA will help to identify the molecular defect in patients with glycogenosis type VII (HMPFK deficiency).