Methylthioadenosine phosphorylase from the archaeon Pyrococcus furiosus. Mechanism of the reaction and assignment of disulfide bonds

European Journal of Biochemistry
Giovanna CacciapuotiMarina Porcelli

Abstract

The extremely heat-stable 5'-methylthioadenosine phosphorylase from the hyperthermophilic archaeon Pyrococcus furiosus was cloned, expressed to high levels in Escherichia coli, and purified to homogeneity by heat precipitation and affinity chromatography. The recombinant enzyme was subjected to a kinetic analysis including initial velocity and product inhibition studies. The reaction follows an ordered Bi-Bi mechanism and phosphate binding precedes nucleoside binding in the phosphorolytic direction. 5'-Methylthioadenosine phosphorylase from Pyrococcus furiosus is a hexameric protein with five cysteine residues per subunit. Analysis of the fragments obtained after digestion of the protein alkylated without previous reduction identified two intrasubunit disulfide bridges. The enzyme is very resistant to chemical denaturation and the transition midpoint for guanidinium chloride-induced unfolding was determined to be 3.0 M after 22 h incubation. This value decreases to 2.0 M in the presence of 30 mM dithiothreitol, furnishing evidence that disulfide bonds are needed for protein stability. The guanidinium chloride-induced unfolding is completely reversible as demonstrated by the analysis of the refolding process by activity assays, ...Continue Reading

References

Feb 20, 1990·Journal of Molecular Biology·S Krishnaswamy, M G Rossmann
Jan 1, 1988·Advances in Experimental Medicine and Biology·Vincenzo ZappiaM Cartenì-Farina
Jan 1, 1972·Methods in Enzymology·K WeberM Osborn
Nov 1, 1969·The Biochemical Journal·A E Pegg, H G Williams-Ashman
Jan 1, 1981·The International Journal of Biochemistry·L ShugartB Chastain
Feb 1, 1982·Biochemical Pharmacology·H G Williams-AshmanP Galletti
Nov 13, 1981·Journal of Chromatography·F Della RagioneVincenzo Zappia
Jul 1, 1994·Bioorganic & Medicinal Chemistry·M W Adams, R M Kelly
Jun 20, 1997·Journal of Molecular Biology·G VogtP Argos
Jul 22, 1998·Advances in Biochemical Engineering/biotechnology·R Ladenstein, G Antranikian
Oct 23, 1998·Extremophiles : Life Under Extreme Conditions·J G ZeikusA Savchenko
Feb 20, 1999·Cell·U JakobJ C Bardwell
Jun 22, 1999·Journal of Molecular Biology·L Xiao, B Honig
Jul 28, 1999·Applied Microbiology and Biotechnology·F NiehausG Antranikian
Apr 25, 2000·Protein Engineering·S KumarR Nussinov
Mar 10, 2001·Microbiology and Molecular Biology Reviews : MMBR·C Vieille, G J Zeikus
Mar 21, 2001·Critical Reviews in Biochemistry and Molecular Biology·R Sterner, W Liebl
May 5, 2001·Pharmacology & Therapeutics·A BzowskaD Shugar
Oct 2, 2001·Cellular and Molecular Life Sciences : CMLS·S Kumar, R Nussinov
Dec 12, 2001·Nature Structural Biology·Einav GrossDeborah Fass
Dec 18, 2001·The Biochemical Journal·Matthew J Pugmire, Steven E Ealick
Jul 11, 2002·Proceedings of the National Academy of Sciences of the United States of America·Parag MallickTodd O Yeates
Jul 20, 1964·Archives of Biochemistry and Biophysics·F SCHLENK, D J EHNINGER
Sep 25, 2003·Extremophiles : Life Under Extreme Conditions·Adriana Molina-HöppnerRudi F Vogel

Citations

Mar 6, 2007·Amino Acids·M A Grillo, S Colombatto
Sep 24, 2011·Archaea : an International Microbiological Journal·Julien Jorda, Todd O Yeates
Aug 23, 2005·PLoS Biology·Morgan BeebyTodd O Yeates
Oct 30, 2007·Biochimica Et Biophysica Acta·Xiusheng ChuDing Li
Mar 8, 2006·The FEBS Journal·Maria G TozziPiero L Ipata
Nov 8, 2016·International Journal of Biological Macromolecules·Ehsan DehnaviKhosro Khajeh
Jan 22, 2019·Critical Reviews in Microbiology·Signe ChristensenWilhelmina M Huston
Jul 29, 2006·The Biochemical Journal·F Grant PearceJuliet A Gerrard
Nov 7, 2019·Biochimica Et Biophysica Acta. Proteins and Proteomics·Sarah KamelAnke Wagner

Related Concepts

5'-Methylthioadenosine phosphorylase
Disulfides
Enzyme Stability
Protein Denaturation
Nicotinamide Riboside Phosphorylase
Fluorescence Spectroscopy
Homologous Sequences, Amino Acid
Protein Folding, Globular
Guanidine Sulfite (1: 1)
Pyrococcus furiosus

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