Biochemistry zyxwvut 1993,32, zyxwvu 13237-1 3244 13237 Site-Directed Mutagenesis of Tyrosine Residues at Nicotinamide Nucleotide Binding Sites of Escherichia coli Transhydrogenase? Torbjiirn Olausson,* Thomas Hultman,g Erland Holmberg,ll and Jan Rydstrbm’sf Department of Biochemistry and Biophysics, University of Gothenburg and Chalmers University of Technology, zyx S-412 96 Gothenburg, Sweden, Department of Biochemistry and Biotechnology, Royal Institute of Technology, S-100 44 Stockholm, Sweden, and Biochemistry Department, Peptide Hormones, Kabi Pharmacia. S- 11 2 87 Stockholm, Sweden Suhail Ahmad,l Natalie A. Glavas,* and Philip D. Braggl Department of Biochemistry, The University of British Columbia, 2146 Health Sciences Mall, Vancouver,British Columbia V6T 123, Canada Received May 7, 1993; Revised Manuscript Received September 2, 1993’ ABSTRACT: Nicotinamide nucleotide transhydrogenase (E.C. 1.6.1.1) from Escherichia coli was investigated with respect to the role of specific conserved tyrosine residues of putative substrate-binding regions. The enzyme from E. coli is made up of two subunits, zyxwvu a (510 residues) and 8 (462 residues). The corresponding enzyme from bovine mitochondria is a single polypeptide (1043 residues) whose N-terminal region zyx corresponds to the a subunit and whose C-terminal region corresponds to the zyxwv , ! 3 subunit. Tyrosines 245 and 1006 of the mitochondrial enzyme have been shown to react selectively with 5’-@-fluorosulfonylbenzoyl)adenosine with inactivation of the enzyme. In E. coli these residues correspond to tyrosine 226 of the a subunit and tyrosine 431 of the B subunit. In addition, tyrosine 315 of the @ subunit is of interest since mutation of an adjacent residue (glycine 314 ) leads to inactivation [Ahmad, S., Glavas, N. A,, & Bragg, P. D. (1992) Eur. J. Biochem. 207, 733-7391. In order to assess the role of the aforementioned conserved tyrosine residues in the mechanism and structure of transhydrogenases, these were replaced by site-specific mutagenesis, using the cloned and overexpressed E. coli transhydrogenase genes [Clarke, D. M., & Bragg, P. D. (1985) J. Bucteriol. 262, 367-3731. Phenylalanine mutants of all three tyrosine residues showed approximately 50% activity or more with regard to catalytic activity assayed as reduction of 3-acetylpyridine-NAD+ by NADPH. These mutants were also active in proton pumping assayed as quenching of 9-methoxy-6-chloro- 2-aminoacridine or quinacrine fluorescence. With respect to catalytic activity these and other mutants were ranked as aY226F > L = H > N, BY431F >> L = H = N = I, and BY315F > N = H > L > D = I = V, indicating that the amino acids at position 226 of the a-subunit and positions 431 and 3 15 of the 8-subunit should be aromatic and sufficiently large and hydrophobic or hydrophilic, but not charged or small (aliphatic) and hydrophobic. These results suggest that tyrosine 226 of the a subunit, and tyrosines 43 1 and 315 of the B subunit are not essential for catalytic activity or proton pumping. The redox-driven proton pump nicotinamide nucleotide transhydrogenase (E.C. 1.6.1.1 .) catalyzes the reversible transfer of a hydride ion between NAD and NADP and the concomitant translocation of n protons across the membrane according to the reaction nH+,,, + NADP’ + NADH * NADPH + NAD’ + nH+i, where “outn and “inwdenote the cytosol and the matrix, respectively, in mitochondria and the periplasmic space and thecytosol, respectively, in bacteria [for reviews, see Rydstrbm (1977), FisherandEarle(1982), Rydstrbmetal. (1987),and Jackson (1991)]. Transhydrogenase is an integral membrane protein which has been purified from several sources, e.&, bovine heart mitochondria (Hiijeberg & Rydstrbm, 1977; Anderson & Fisher, 1978; Wu et al., 1982; Persson et al., 1984; Phelps & Hatefi, 1984) and Escherichia coli (Clarke f This work was supported by the Swedish Natural Science Research Council and the Medical Research Council of Canada. N.A.G. acknowl- edges the award of a MRC Studentship. * To whom correspondence should be addressed. t University of Gothenburg and Chalmers University of Technology. 8 Royal Institute of Technology. 11 Kabi Pharmacia. zyxwvutsrq 1 The University of British Columbia. zyxwvutsrq e Abstract published in Advance ACS Abstracts, October 15, 1993. 0006-2960/93/0432-13237$04.00/0 & Bragg, 1985), and characterized extensively in the recon- stituted form with and without other proton pumps in phospholipid vesicles (Wu et al., 1982; Persson et al,, 1984; Clarke & Bragg, 1985a; Earle & Fisher, 1979, 1980; Pennington et al., 1981; Eytan et al., 1987a,b, 1990). By employment of reconstituted vesicles, the number n in the above reaction has been concluded to be 1 [cf. Rydstrbm et al. (1987); see also Olausson et al. (1992) and Hoek and RydstrBm (1988)l. However, in chromatophores from Rho- dospirillum rubrum, n has been shown to be 0.5 (Bizouam & Jackson, 1993). The active bovine heart enzyme is a homodimer with a molecular mass of the monomer of about 109 kDa (Wu & Fisher, 1983; Persson et al., 1987; Ormb et al., 1992), whereas the E. coli enzyme has 2 subunits and a composition of a282 in the active enzyme (Hou et al., 1990). The genes for the E. coli transhydrogenase (Clarke & Bragg, 1985a; Clarke et al., 1986) and the cDNA for bovine heart mitochondrial enzyme (Yamaguchi et al., 1988) have been cloned and sequenced. However, even though the complete bovine transhydrogenase gene has been synthesized from partial clones (Holmberg et al., 1992), only the genes for the a and subunits of E. coli transhydrogenase have been expressed individually (Clarke & Bragg, 1986) or simulta- neously (Clarke & Bragg, 1985b),using various E. colistrains as host cells. This expression represents approximately a 70- zyxwvutsrqp 0 1993 American Chemical Society