SITE-DIRECTED MUTAGENESIS OF TYROSINE RESIDUES AT NICOTINAMIDE NUCLEOTIDE-BINDING SITES OF ESCHERICHIA-COLI TRANSHYDROGENASE

Nicotinamide nucleotide transhydrogenase (E.C. 1.6.1.1) from Escherich ia coli was investigated with respect to the role of specific conserve d tyrosine residues of putative substrate-binding regions. The enzyme from E. coli is made up of two subunits, alpha (510 residues) and beta (462 residues). The corresponding enzyme from bovine mitochondria is a single polypeptide (1043 residues) whose N-terminal region correspon ds to the alpha subunit and whose C-terminal region corresponds to the beta subunit. Tyrosines 245 and 1006 of the mitochondrial enzyme have been shown to react selectively with 5'-(p-fluorosulfonylbenzoyl)aden osine with inactivation of the enzyme. In E. coli these residues corre spond to tyrosine 226 of the alpha subunit and tyrosine 431 of the bet a subunit. In addition, tyrosine 315 of the beta subunit is of interes t since mutation of an adjacent residue (glycine 314) leads to inactiv ation [Ahmad, S., Glavas, N. A., & Bragg, P. D. (1992) Eur. J. Biochem . 207, 733-739]. In order to assess the role of the aforementioned con served tyrosine residues in the mechanism and structure of transhydrog enases, these were replaced by site-specific mutagenesis, using the cl oned and overexpressed E. coli transhydrogenase genes [Clarke, D. M., & Bragg, P. D. (1985) J. Bacteriol. 162, 367-373]. Phenylalanine mutan ts of all three tyrosine residues showed approximately 50% activity or more with regard to catalytic activity assayed as reduction of 3-acet ylpyridine-NAD+ by NADPH. These mutants were also active in proton pum ping assayed as quenching of 9-methoxy-6-chloro-2-aminoacridine or qui nacrine fluorescence. With respect to catalytic activity these and oth er mutants were ranked as alphaY226F > L = H > N, betaY431F >> L = H = N = I, and betaY315F > N = H > L > D = I = V, indicating that the ami no acids at position 226 of the alpha-subunit and positions 431 and 31 5 of the beta-subunit should be aromatic and sufficiently large and hy drophobic or hydrophilic, but not charged or small (aliphatic) and hyd rophobic. These results suggest that tyrosine 226 of the alpha subunit , and tyrosines 431 and 315 of the beta subunit are not essential for catalytic activity or proton pumping.