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Searchgui enzyme definition12/29/2022 ![]() Isoform (Uniprot)ĬtHTM1P (the product of the gene CTHT_0058730, Uniprot entryĥ0−1092-pHLsec vector for secreted mammalian cell expression is described in detail in the Open Laboratory Notebook page (seeĮxtended data 18). HsGAPDH structure determined to date (1.52 Å). HsGAPDH crystal structures carrying a Cysteine-S-sulfonic acid modification of the catalytic cysteine Cys152. HsGAPDH monoclinic polymorphs and are the first HsGAPDH crystal structures we describe (which we label as P2 ![]() A fraction of the molecules shows Cys-S-sulfonic acid instead of Cys in the active site. HsGAPDH purified from the supernatant of a HEK293F cell culture. In this paper, we present and discuss mass spectrometry and crystallographic evidence supporting partial oxidation of the catalytic Cys residue of Cys oxidised modifications and GAPDH PDB entries that carry them at the catalytic Cys152.Īka Cysteine sulfenic acid (CSX, PDB ID 1J0X) Īka 3-Sulfino-L-alanine (CSD, PDB IDs 2VYN and 2VYV) Ĭ: S-hydroxy-cysteine (CSO, not described in any GAPDH structures) ĭ: Cysteine-S-sulfonic acid (CSU, PDB ID 5M6D and this work) Cysteine-S-sulfinic acid (CSD, seeįigure 1B) has been observed in structures of rat GAPDHS andįigure 1D) modifies the catalyic Cys in a structure ofġ7. ![]() In particular, cysteine sulfenic acid (CSX, seeįigure 1A) has been observed in the structure of rabbit muscle GAPDHġ5 (of course, by X-ray diffraction alone, this modification may be difficult to distinguish from S-hydroxy-cysteine (CSO, seeįigure 1C)). A number of studies have reported the GAPDH catalytic Cys carrying oxidised post-translational modifications. GAPDH was one of the first enzymes to be crystallisedġ2 and one of the first enzymes whose structure was determined by X-ray crystallographyġ4. The latter enables association of GAPDHS to the sperm flagellum fibrous sheath, so that the enzyme – together with other glycolytic enzymes - provides a localised source of ATP that is essential for sperm motility In multiple mammalian species the sperm isozyme (GAPDHS) shares about 70% amino acid identity with the somatic isozyme, and possesses an additional 72-residues N-terminal extension. HsGAPDHS (G3PT_HUMAN (Uniprot O14556)), which is expressed only in the post-meiotic period of spermatogenesis. ![]() HsGAPDH isoform, the human genome encodes a testis-specific isoform Several studies indicate that the enzyme has pleiotropic functions independent of its canonical role in glycolysis The GAPDH-catalysed forward reaction occurs at an important transition point in glycolysis between the enzymatic steps that consume and generate ATP. GAPDH is essential for glycolysis and gluconeogenesis it catalyses the NAD+-dependent oxidative phosphorylation of n-glyceraldehyde-3-phosphate to 1,3- diphospho-n-glycerate (and its reverse reaction)Ħ. In the course of a research effort aimed at the purification of recombinantĬtHTM1P from the supernatant of HEK293F cells, we purified, crystallised and determined crystal structures of human n-Glyceraldehyde-3-phosphate dehydrogenase ( Of course, in absence of secretion of the desired recombinant protein at hand, contaminants are the only proteins present in recombinant protein expression systemsĤ. They owe their popularity to ease of handling, robust growth rate, excellent transfectability, high capacity for recombinant protein expression, low-cost media requirements and low levels of secreted contaminantsĢ. Mammalian HEK293F cells are routinely used in conjunction with secreted protein expression vectors for recombinant protein productionġ. ![]()
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