THE ROLE OF CONSERVED HISTIDINE-RESIDUES IN THE PYRIDINE-NUCLEOTIDE TRANSHYDROGENASE OF ESCHERICHIA-COLI

The pyridine nucleotide transhydrogenase of Escherichia coli catalyzes the reversible transfer of hydride ion equivalents between NAD(+) and NADP(+), coupled to translocation of protons across the cytoplasmic m embrane. The role of histidine residues in catalysis was investigated by chemical modification with diethylpyrocarbonate and by site-directe d mutagenesis. Diethylpyrocarbonate inhibited both hydride ion transfe r and coupled proton translocation. Histidine residues were modified a s shown spectroscopically and by the ability of hydroxylamine to cause reversal of inhibition. Complete inhibition of hydride ion transfer o ccurred following modification of 10 residues/enzyme molecule. Site-di rected mutagenesis of single conserved histidine residues or the prese nce of substrates did not provide resistance to inhibition by diethylp yrocarbonate. It is concluded that diethylpyrocarbonate inhibition was a consequence of the structural changes brought about by modification of many histidine residues. With the exception of beta-subunit residu e His91 (beta His91), in which mutation can result in specific loss of proton translocation activity [Glavas, N. A., Hou, C. & Bragg, P. D. (1995) Biochemistry 34, 7694-7702], site-directed mutation of the rema ining conserved residues alpha His450, beta His161, beta His345 and be ta His354 did not demonstrate a direct role for these residues in cata lysis. Mutation of beta His161 had relatively little effect on the pro perties of the enzyme. By contrast, mutation of alpha His450, beta His 345 and beta His354 caused major loss of enzyme activities which was p robably due to alterations in the structure of the enzyme. These alter ations were reflected in changes in the K-m values for transhydrogenat ion.