IDENTIFICATION OF N,N'-DICYCLOHEXYLCARBODIIMIDE-REACTIVE GLUTAMIC ANDASPARTIC-ACID RESIDUES IN ESCHERICHIA-COLI TRANSHYDROGENASE AND THE EXCHANGE OF THESE BY SITE-SPECIFIC MUTAGENESIS

Pyridine nucleotide transhydrogenase (EC 1.6.1.1) from Escherichia col i was investigated with respect to the role of glutamic and aspartic a cid residues reactive to N,N'-dicyclohexylcarbodiimide (DCCD) and pote ntially involved in the proton-pumping mechanism of the enzyme. The E. coli transhydrogenase consists of an alpha (510 residues) and a beta (462 residues) subunit. DCCD reacts with the enzyme to inhibit catalyt ic activity and proton pumping. This reagent modifies Asp(alpha232), G lu(alpha238), and Glu(alpha240) as well as amino acid residue(s) in th e beta subunit. Using the cloned and overexpressed E. coli transhydrog enase genes (Clarke, D. M., and Bragg, P. D. (1985) J. Bacteriol. 162, 367-373), Asp(alpha232) and Glu(alpha238) were replaced independently by site-specific mutagenesis. In addition, Asp(alpha232), Glu(alpha23 8), and Glu(alpha240) were replaced to generate triple mutants. The sp ecific catalytic activities of the mutant transhydrogenases alphaD232N , alphaD232E, alphaD232K, alphaD232H, alphaE238K, and alphaE238Q as we ll as of the triple mutants alphaD232N,alphaE238Q,alphaE240Q and alpha D232H,alphaE238Q, alphaE240Q were in the range of 40-90% of the wild-t ype activity. Proton-pumping activity was present in all mutants. Exam ination of the extent of subunit modification by [C-14]DCCD revealed t hat the label was still incorporated into both alpha and beta subunits in the Asp(alpha232) mutants, but that the alpha subunit was not labe led in the triple mutants. Catalytic and proton-pumping activities wer e nearly insensitive to DCCD in the triple mutants. This suggests that loss of catalytic and proton pumping activities is associated with mo dification of the aspartic and glutamic acid residues of the a subunit . In the presence of the substrate NADPH, the rate of modification of the beta subunit by [C-14]DCCD was increased, and there was a greater extent of enzyme inactivation. By contrast, NADH and 3-acetylpyridine- NAD+ protected the catalytic activity of the transhydrogenase from inh ibition by DCCD. The protection was particularly marked in the E238Q a nd E238K mutants. It is concluded that the Asp(alpha232), Glu(alpha238 ), and Glu(alpha240) residues are not essential for catalytic activity or proton pumping. The inactivation by DCCD is likely due to the intr oduction of a sterically hindering group that reacts with the identifi ed acidic residues close to the NAD(H)-binding site.