Residue β-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)Nature370, 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, β-L390F, was studied in the presence of various replacements at position 198.In vivoandin vitroanalyses 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 β-subunit is not stablein vivowith Gly at position 198. These results indicate that V198 forms critical hydrophobic interactions with the adenine ring of the nucleotide.