Mutagenesis of beta-V198 in the F1-ATPase of yeast Saccharomyces cerevisiae and its role in binding nucleotide.
Residue beta-V198 of the yeast mitchondrial F1-ATPase abuts the P-loop motif and the side chain is within 3.8 A of the nucleotide as shown in the crystal structure of the bovine ATPase [J. P. Abrahams, A. G. W. Leslie, R. Lutter, and J. E. Walker (1984) Nature 370,621-628]. This study has made and analyzed 17 replacements of V198 to understand the importance of the side chain in the nucleotide binding site. In addition, a suppressor of V198S, beta-L390F, was studied in the presence of various replacements at position 198. In vivo and in vitro analyses indicate that the Val side chain is critical for forming a stable and active enzyme. Biochemical analysis of mitochondria isolated from the mutant strains indicates that amino acids with hydrophobic side chains are the most effective replacements. In addition, size is important, but a large side chain can be largely compensated for until the size reaches that of the Phe and Trp. A methyl group is the minimal side chain necessary for function, as the beta-subunit is not stable in vivo with Gly at position 198. These results indicate that V198 forms critical hydrophobic interactions with the adenine ring of the nucleotide.
Sosa-Peinado
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Department of Biological Chemistry, The Chicago Medical School, North Chicago, Illinois 60064, USA.
Mueller
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DM
D000225:Adenine / Q000737:chemistry; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001386:Azides / Q000494:pharmacology; D001665:Binding Sites; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009840:Oligomycins / Q000494:pharmacology; D010455:Peptides; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D012995:Solubility; D013329:Structure-Activity Relationship
D000900:Anti-Bacterial Agents; D001386:Azides; D004791:Enzyme Inhibitors; D009840:Oligomycins; D010455:Peptides; C012953:efrapeptin; D006180:Proton-Translocating ATPases; D000225:Adenine