Active site complementation in engineered heterodimers of Escherichia coli glutathione reductase created in vivo.
By directed mutagenesis of the cloned Escherichia coli gor gene encoding the dimeric flavoprotein glutathione reductase, Cys-47 (a cysteine residue forming an essential charge-transfer complex with enzyme-bound FAD) was converted to serine (C47S) and His-439 (required to facilitate protonation of the reduced glutathione) was converted to glutamine (H439Q). Both mutant genes were placed in the same plasmid, pHD, where each of them came under the control of a strong tac promoter. This was designed to achieve equal over-expression of both genes in the same E. coli cell. The parental homo-dimers show no (C47S) or very little (H439Q) activity as glutathione reductases. The formation in vivo of heterodimers, carrying one crippled and one fully functional active site, was detected by absorbance spectroscopy and fluorescence emission spectrometry of enzyme-bound FAD and by active site complementation. The fractional distribution of homo- and hetero-dimers was in accord with that expected for a random association of enzyme subunits. In a homo-dimer, the H439Q mutation leads to a big fall in the value of Km for NADPH which binds some 1.8 nm from the point of mutation (Berry, A., Scrutton, N.S. & Perham, R. N. Biochemistry 28, 1264-1269 (1989)). However, the one active site in the H439Q/C47S hetero-dimer exhibited kinetic parameters similar to those of the wild-type enzyme. Thus, the effect of the H439Q mutation must be retained within the active site that accommodates it and is not transmitted through the protein to the second active site across the subunit interface.(ABSTRACT TRUNCATED AT 250 WORDS)
Scrutton
N S
NS
Cambridge Centre for Molecular Recognition, Department of Biochemistry, University of Cambridge, U.K.
Berry
A
A
Deonarain
M P
MP
Perham
R N
RN
D000595:Amino Acid Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D005980:Glutathione Reductase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D015202:Protein Engineering
D005980:Glutathione Reductase