An unexpected phosphate binding site in Glyceraldehyde 3-Phosphate Dehydrogenase: Crystal structures of apo, holo and ternary complex of Cryptosporidium parvum enzyme

Cook, William J.; Senkovich, Olga; Chattopadhyay, Debasish
January 2009
BMC Structural Biology;2009, Vol. 9, Special section p1
Academic Journal
Background: The structure, function and reaction mechanism of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) have been extensively studied. Based on these studies, three anion binding sites have been identified, one 'Ps' site (for binding the C-3 phosphate of the substrate) and two sites, 'Pi' and 'new Pi', for inorganic phosphate. According to the original flip-flop model, the substrate phosphate group switches from the 'Pi' to the 'Ps' site during the multistep reaction. In light of the discovery of the 'new Pi' site, a modified flip-flop mechanism, in which the C-3 phosphate of the substrate binds to the 'new Pi' site and flips to the 'Ps' site before the hydride transfer, was proposed. An alternative model based on a number of structures of B. stearothermophilus GAPDH ternary complexes (non-covalent and thioacyl intermediate) proposes that in the ternary Michaelis complex the C-3 phosphate binds to the 'Ps' site and flips from the 'Ps' to the 'new Pi' site during or after the redox step. Results: We determined the crystal structure of Cryptosporidium parvum GAPDH in the apo and holo (enzyme + NAD) state and the structure of the ternary enzyme-cofactor-substrate complex using an active site mutant enzyme. The C. parvum GAPDH complex was prepared by pre-incubating the enzyme with substrate and cofactor, thereby allowing free movement of the protein structure and substrate molecules during their initial encounter. Sulfate and phosphate ions were excluded from purification and crystallization steps. The quality of the electron density map at 2Ã… resolution allowed unambiguous positioning of the substrate. In three subunits of the homotetramer the C-3 phosphate group of the noncovalently bound substrate is in the 'new Pi' site. A concomitant movement of the phosphate binding loop is observed in these three subunits. In the fourth subunit the C-3 phosphate occupies an unexpected site not seen before and the phosphate binding loop remains in the substrate-free conformation. Orientation of the substrate with respect to the active site histidine and serine (in the mutant enzyme) also varies in different subunits. Conclusion: The structures of the C. parvum GAPDH ternary complex and other GAPDH complexes demonstrate the plasticity of the substrate binding site. We propose that the active site of GAPDH can accommodate the substrate in multiple conformations at multiple locations during the initial encounter. However, the C-3 phosphate group clearly prefers the 'new Pi' site for initial binding in the active site.


Related Articles

  • The composition of phosphate granules in the digestive glands of marine prosobranch gastropods: variation in relation to taxonomy. GIBBS, P. E.; NOTT, J. A.; NICOLAIDOU, A.; BEBIANNO, M. J. // Journal of Molluscan Studies;Nov1998, Vol. 64 Issue 4, p423 

    The composition of some 1150 phosphate granules in the digestive glands of over 40 species of marine prosobranch gastropods has been surveyed using a simple preparation technique and semi-quantitative SEM x-ray microanalysis. Spectral peaks for Mg, K, Ca, Mn, Fe and Zn were compared to that of...

  • Bacterial community dynamics in the hyporheic zone of an intermittent stream. Febria, Catherine M; Beddoes, Paul; Fulthorpe, Roberta R; Williams, D Dudley // ISME Journal: Multidisciplinary Journal of Microbial Ecology;May2012, Vol. 6 Issue 5, p1078 

    The dynamics of in situ bacterial communities in the hyporheic zone of an intermittent stream were described in high spatiotemporal detail. We assessed community dynamics in stream sediments and interstitial pore water over a two-year period using terminal-restriction fragment length...

  • ADP.  // Taber's Cyclopedic Medical Dictionary (2009);2009, Issue 21, p51 

    An encyclopedia entry for "ADP," which refers to adenosine diphosphate, is presented.

  • How Actin Filaments Doff Their Pi Cap. Sedwick, Caitlin // PLoS Biology;Sep2011, Vol. 9 Issue 9, p1 

    The article focuses on a study on the release of phosphate molecule called Pi from adenosine diphosphate (ADP)-Pi-actin and the polymerization and depolymerization of actin filaments. It says that an apparatus which uses microfluidics in releasing free actin from a chamber with actin filaments...

  • Convenient, tandem and one-reaction vessel synthesis of mixed dialkylated 2-naphthols from 2-tetralone. Jha, Amitabh; Dimmock, Jonathan R. // Canadian Journal of Chemistry;Apr2003, Vol. 81 Issue 4, p293 

    A convenient acid-catalyzed method for the synthesis of 1-alkyl-2-alkoxynaphthalenes by one-reaction vessel mixed alkylation of 2-tetralone, using aryl or alkyl aldehydes and alcohols followed by aromatization, is described.

  • Oxidation of 1-Oxa-5-azaspiro[5.5]undecane with cyclohexanone. Kukharev, B. F.; Stankevich, V. K.; Klimenko, G. R.; Bayandin, V. V.; Kukhareva, V. A. // Russian Journal of Organic Chemistry;Mar2008, Vol. 44 Issue 3, p477 

    The article discusses a study on the oxidation of 1-oxa-5-azaspirol undecane with cyclohexanone to produce 5,6,7,8-tetrahydroquinoline as an example of synthesis of pyridine derivatives via oxidation of perhydro-1,3-oxazines. The oxidation of oxazolidines with carbonyl compounds, initial...

  • Mild and Efficient Strontium Chloride Hexahydrate-Catalyzed Conversion of Ketones and Aldehydes into Corresponding gem- Dihydroperoxides by Aqueous H2O2. Azarifar, Davood; Khosravi, Kaveh; Soleimanei, Fatemeh // Molecules;Mar2010, Vol. 15 Issue 3, p1433 

    SrCl2·6H2O has been shown to act as an efficient catalyst for the conversion of aldehydes or ketones into the corresponding gem-dihydroperoxides (DHPs) by treatment with aqueous H2O2 (30%) in acetonitrile. The reactions proceed under mild and neutral conditions at room temperature to afford...

  • Perfluoroalkanesulfonamide Organocatalysts for Asymmetric Conjugate Additions of Branched Aldehydes to Vinyl Sulfones. Kosuke Nakashima; Murahashi, Miho; Hiroki Yuasa; Mariko Ina; Tada, Norihiro; Itoh, Akichika; Hirashima, Shin-ichi; Yuji Koseki; Miura, Tsuyoshi // Molecules;Dec2013, Vol. 18 Issue 12, p14529 

    Asymmetric conjugate additions of branched aldehydes to vinyl sulfones promoted by sulfonamide organocatalyst 6 or 7 have been developed, allowing facile synthesis of the corresponding adducts with all-carbon quaternary stereocenters in excellent yields with up to 95% ee.

  • N,N-Dimethylpyridin-4-Amine Mediated Protocol for Cyanoethoxycarbonylation of Aldehydes Under Solvent-Free Conditions. Khan, Noor-ul H.; Agrawal, Santosh; Kureshy, Rukhsana I.; Bera, Prasanta K.; Abdi, Sayed H. R.; Bajaj, Hari C. // Catalysis Letters;Jul2010, Vol. 137 Issue 3/4, p255 

    N,N-Dimethylpyridin-4-amine (DAMP) (10 mol%) was successfully employed as catalyst for cyanoethoxycarbonylation of aromatic and aliphatic aldehydes at room temperature under solvent free condition to give corresponding ethylcyanocarbonates in excellent isolated yield (up to 95%) in 15–90...


Read the Article


Sorry, but this item is not currently available from your library.

Try another library?
Sign out of this library

Other Topics