AN ATTEMPT TO CONVERT NONCATALYTIC NUCLEOTIDE-BINDING SITE OF F1-ATPASE TO THE CATALYTIC SITE - HYDROLYSIS OF TETHERED ATP BY MUTATED ALPHA-SUBUNITS IN THE ENZYME

The alpha and beta subunits of F-1-ATPase are homologous in primary st ructure and have similar folding topologies. The position of the essen tial Glu residue in the catalytic sites which reside in the alpha subu nits is occupied by a Gin residue in the noncatalytic nucleotide bindi ng sites which reside in the alpha subunits. To test if an exchange of catalytic and noncatalytic binding sites is possible, we have replace d the Gln-Lys sequence in the noncatalytic binding site of the alpha s ubunit with Glu-Arg and, reciprocally, the Glu in the catalytic site o f the beta subunit with Gin. The resultant mutant alpha(3) beta(3) gam ma complex lost steady-state ATPase activity. However, HPLC analysis o f tryptic digests of the mutant alpha(3) beta(3) gamma complex which h ad been photolabeled with 2-N-3-[8-H-3]ATP revealed that ATP tethered to the noncatalytic binding site was hydrolyzed, indicating that a pri mitive catalytic ability was generated at the alpha subunit by the int roduced Glu. (C) 1996 Academic Press, Inc.