EFFECT OF TRUNCATION AND MUTATION OF THE CARBOXYL-TERMINAL REGION OF THE BETA-SUBUNIT ON MEMBRANE ASSEMBLY AND ACTIVITY OF THE PYRIDINE-NUCLEOTIDE TRANSHYDROGENASE OF ESCHERICHIA-COLI

The pyridine nucleotide transhydrogenase of Escherichia coil is a prot on pump composed of two different subunits (alpha and beta) assembled as a tetramer (alpha 2 beta 2) in the cytoplasmic membrane. A series o f mutants was generated in which the carboxyl-terminal region of the b eta subunit was progressively truncated. Removal of the two carboxyl-t erminal amino acid residues prevented incorporation of the enzyme into the cytoplasmic membrane. Deletion of the carboxyl-terminal amino aci d allowed incorporation of the a subunit to near normal levels, but th e amount of the beta subunit was much decreased. It is concluded that, although the a subunit can be incorporated into the cytoplasmic membr ane without the beta subunit, the carboxyl-terminal region of the beta subunit is involved in determining the correct conformation of the al pha subunit for assembly. The carboxyl-terminal amino acid of the beta subunit, beta Leu462, and the penultimate residue, beta Ala461, were individually mutated and the effect on two transhydrogenase activities determined. The reduction of 3-acetylpyridine adenine dinucleotide (A cPyAD(+)) by NADPH, and by NADH in the presence of NADP(+), was decrea sed maximally by about 60%. The reduction of AcPyAD(+) by NADH in the absence of NADP(+) was decreased to a greater extent. Most mutants of beta Leu462 showed at least an 80% reduction in activity as well as ab normal kinetics. The abnormal kinetics were explored in the beta A461P mutant and were attributed to tighter binding of the product AcPyADH. This compound competed with NADP(+) at the NADP(H)-binding site. It i s concluded that the carboxyl-terminal region of the beta subunit cont ributes to the NADP(H)-binding site on this subunit (C) 1998 Elsevier Science B.V. All rights reserved.