KINETIC RESOLUTION OF THE REACTION CATALYZED BY PROTON-TRANSLOCATING TRANSHYDROGENASE FROM ESCHERICHIA-COLI AS REVEALED BY EXPERIMENTS WITHANALOGS OF THE NUCLEOTIDE SUBSTRATES

The mechanism, by which transhydrogenase couples transfer of H- equiva lents between NAD(H) and NADP(H) to the translocation of protons acros s a membrane, has been investigated in the solubilised, purified enzym e from Escherichia coli using analogues of the nucleotide substrates. The key observation was that, at low pH and ionic strength, solubilise d transhydrogenase catalysed the very rapid reduction of acetylpyridin e adenine dinucleotide (an analogue of NAD(+)) by NADH, but only in th e presence of either NADP(+) or NADPH. This indicates that the rates o f release of NADP(+) and NADPH from their binary complexes with the en zyme are slow. The dependences on pH and salt concentration suggest th at (a) release of both NADP(+) and NADPH are accompanied by the releas e of H+ from the enzyme and (b) increased ionic strength decreases the value of the pK(a) of the group responsible for H+ release. Modificat ion of the enzyme with N,N-1-dicyclohexylcarbodiimide led to inhibitio n of the rate of release of NADP(+) and NADPH from the enzyme, but had a much smaller effect on the binding and release of NAD(+), NADH and their analogues and on the interconversion of the ternary complexes of the enzyme with its substrates. It is considered that the binding and release of H+, which accompany the binding and release of NADP(+)/NAD PH, might be central to the mechanism of proton translocation by the e nzyme in its membrane-bound state.