tailieunhanh - Báo cáo khoa học: Cd2+-induced aggregation of Escherichia coli pyrophosphatase

We report here thatEscherichia coli pyrophosphatase aggregates in the presence of millimolar Cd 2+ . This highly cooperative process was specific to both the metal ion and the protein and could be reversed fully by decreasing the Cd 2+ concentration. Aggregation was enhanced by Mg 2+ , the natural cofactor of pyrophosphatase, and Mn 2+ . Mutations at the intersubunit metal-binding site had no effect, whereas mutation at Glu139, which is part of the peripheral metal-binding site found in pyrophosphatase crystals near the contact region between two enzyme mole-cules, suppressed aggregation. These findings indicate that aggregation is affected by Cd 2+ binding to the peripheral metal-binding site, probably by strengthening intermole-cular Trp149–Trp149¢stacking interactions | Eur. J. Biochem. 271 3064-3067 2004 FEBS 2004 doi Cd2 -induced aggregation of Escherichia coli pyrophosphatase Yury V. Zimenkov1 Anu Salminen2 Irina S. Efimova1 Reijo Lahti2 and Alexander A. Baykov1 1A. N. Belozersky Institute of Physico-Chemical Biology and School of Chemistry Moscow State University Moscow Russia department of Biochemistry University of Turku Finland We report here that Escherichia coli pyrophosphatase aggregates in the presence of millimolar Cd2 . This highly cooperative process was specific to both the metal ion and the protein and could be reversed fully by decreasing the Cd2 concentration. Aggregation was enhanced by Mg2 the natural cofactor of pyrophosphatase and Mn2 . Mutations at the intersubunit metal-binding site had no effect whereas mutation at Glu139 which is part of the peripheral metal-binding site found in pyrophosphatase crystals near the contact region between two enzyme molecules suppressed aggregation. These findings indicate that aggregation is affected by Cd2 binding to the peripheral metal-binding site probably by strengthening intermole-cular Trp149-Trp149 stacking interactions. Keywords aggregation cadmium inorganic pyrophosphatase site-directed mutagenesis. Protein aggregation is a common phenomenon with important practical implications. A variety of diseases including the amyloidoses and prion diseases as well as other protein deposition disorders involve protein aggregation 1 . In most cases the proteins that aggregate are totally or partially unfolded and the aggregation which occurs via hydrophobic interactions is almost completely irreversible 2 3 . Examples of proteins aggregating in their native state other than salting out and isoelectric point precipitation are less common with the aggregation of the mutant hemoglobin that causes sickle-cell anemia being the best known example 4 . In addition Zn2 and other divalent cations have been reported to aggregate native dodecameric .