IDENTIFICATION OF AN ASPARTIC-ACID RESIDUE IN THE BETA-SUBUNIT WHICH IS ESSENTIAL FOR CATALYSIS AND PROTON-PUMPING BY TRANSHYDROGENASE FROMESCHERICHIA-COLI

Based on the alignment of 7 known amino acid sequences, including the recently determined sequences for the mouse and human enzymes, a highl y conserved acidic domain was identified which in the Escherichia coli enzyme is located close to the C-terminal end of the predicted NADP(H )-binding site of the beta subunit. The effect of replacing the four c onserved acidic residues, beta E361, beta E374, beta D383 and beta D39 2, in this domain on catalytic and proton-pumping activity was tested by site-directed mutagenesis. In addition, beta E371, which is not con served but located in the same domain, was also mutated. Of these resi dues, beta Asp 392 proved to be the only residue which is essential fo r both activities. However, two beta Asp 392 mutants were still partly active in catalyzing the cyclic reduction of 3-acetylpyridine-NAD(+) by NADH in the presence of NADPH, suggesting that the mutations did no t cause a global change but rather a subtle local change influencing t he dissociation of NADP(H). It is proposed that beta Asp 392 together with the previously identified beta His91 form part of a proton wire i n transhydrogenase.