Effect of truncation and mutation of the carboxyl-terminal region of the β subunit on membrane assembly and activity of the pyridine nucleotide transhydrogenase of Escherichia coli

Abstract

The pyridine nucleotide transhydrogenase of Escherichia coli is a proton pump composed of two different subunits (α and β) assembled as a tetramer (α 2β 2) in the cytoplasmic membrane. A series of mutants was generated in which the carboxyl-terminal region of the β subunit was progressively truncated. Removal of the two carboxyl-terminal amino acid residues prevented incorporation of the enzyme into the cytoplasmic membrane. Deletion of the carboxyl-terminal amino acid allowed incorporation of the α subunit to near normal levels, but the amount of the β subunit was much decreased. It is concluded that, although the α subunit can be incorporated into the cytoplasmic membrane without the β subunit, the carboxyl-terminal region of the β subunit is involved in determining the correct conformation of the α subunit for assembly. The carboxyl-terminal amino acid of the β subunit, βLeu462, and the penultimate residue, βAla461, were individually mutated and the effect on two transhydrogenase activities determined. The reduction of 3-acetylpyridine adenine dinucleotide (AcPyAD +) by NADPH, and by NADH in the presence of NADP +, was decreased maximally by about 60%. The reduction of AcPyAD + by NADH in the absence of NADP + was decreased to a greater extent. Most mutants of βLeu462 showed at least an 80% reduction in activity as well as abnormal kinetics. The abnormal kinetics were explored in the βA461P mutant and were attributed to tighter binding of the product AcPyADH. This compound competed with NADP + at the NADP(H)-binding site. It is concluded that the carboxyl-terminal region of the β subunit contributes to the NADP(H)-binding site on this subunit