MUTATIONAL ANALYSIS OF THE FIRST EXTRACELLULAR LOOP REGION OF THE H-ATPASE FROM SACCHAROMYCES-CEREVISIAE()

Transmembrane segments 1 and 2 of the yeast plasma membrane H+-ATPase are believed to form a helical hairpin structure that is joined by a s hort extracytoplasmic loop. The hairpin head region (Ala(135)-Phe(144) ) was probed using site-directed mutagenesis, Scanning alanine mutagen esis produced functional H+-ATPase at all positions except Leu(138), A sp(143), and Phe(144). D140A and V142A gave strong hygromycin B resist ance and low pH sensitivity suggesting a major kinetic defect in these mutant enzymes. Other amino acid substitutions, such as L138Y, were h ighly perturbing, while mutations S139E and D140E produced minor effec ts on phenotype. Small uncharged residues Gly and Ala, which were inse rted between Leu(138) and Ser(139) to examine the importance of loop l ength on Ht-ATPase function, were well tolerated, while the insertion of a polar Ser residue was highly perturbing. Other additions were not tolerated by the enzyme. These results suggest that the turn region h as limited structural flexibility. The conserved Phe(144) residue coul d be changed to Trp with a minor effect on phenotype. However, neither Tyr,Arg, nor small hydrophobic residues could substitute, suggesting that this region is closely packed and hydrophobic. ATP hydrolysis mea surements showed that V-max was significantly reduced in nearly all. m utant enzymes, except D140E; whereas, K-m values were nearly normal. V anadate-sensitivity and pH profiles for ATP hydrolysis were nearly nor mal for all mutant enzymes except insertion mutant S138+. Mutants with extreme phenotypes (S138+, Tyr(138)) showed significantly altered med ium acidification profiles. These results support the notion that the hairpin head region linking transmembrane segments 1 and 2 forms a tig htly packed conformationally sensitive domain that is coupled to the c atalytic ATP hydrolysis domain.