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 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, alpha (510 residues) and beta (462 residues). The corresponding enzyme from bovine mitochondria is a single polypeptide (1043 residues) whose N-terminal region corresponds 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)adenosine with inactivation of the enzyme. In E. coli these residues correspond to tyrosine 226 of the alpha subunit and tyrosine 431 of the beta subunit. In addition, tyrosine 315 of the beta 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, 733-739]. 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. Bacteriol. 162, 367-373]. 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Olausson
T
T
Department of Biochemistry and Biophysics, University of Gothenburg, Sweden.
Hultman
T
T
Holmberg
E
E
Rydström
J
J
Ahmad
S
S
Glavas
N A
NA
Bragg
P D
PD
D000595:Amino Acid Sequence; D001665:Binding Sites; D001693:Biological Transport, Active; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D009250:NADP Transhydrogenases / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000737:chemistry
D009243:NAD; D014443:Tyrosine; D009250:NADP Transhydrogenases