Methylthioadenosine phosphorylase from the archaeon Pyrococcus furiosus. Mechanism of the reaction and assignment of disulfide bonds.
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, fluorescence measurements and SDS/PAGE. The finding of multiple disulfide bridges in 5'-methylthioadenosine phosphorylase from Pyrococcus furiosus argues strongly that disulfide bond formation may be a significant molecular strategy for stabilizing intracellular hyperthermophilic proteins.
Cacciapuoti
Giovanna
G
Dipartimento di Biochimica e Biofisica F. Cedrangolo, Seconda Università di Napoli, Naples, Italy. giovanna.cacciapuoti@unina2.it
Moretti
Maria Angela
MA
Forte
Sabrina
S
Brio
Assunta
A
Camardella
Laura
L
Zappia
Vincenzo
V
Porcelli
Marina
M
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004220:Disulfides / Q000737:chemistry*; D004795:Enzyme Stability; D019791:Guanidine; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D017510:Protein Folding; D011683:Purine-Nucleoside Phosphorylase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D020027:Pyrococcus furiosus / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence
D004220:Disulfides; D011683:Purine-Nucleoside Phosphorylase; C021784:5'-methylthioadenosine phosphorylase; D019791:Guanidine