Active site complementation in engineered heterodimers of Escherichia coli glutathione reductase created in vivo

摘要: By directed mutagenesis of the cloned Escherichia coli gor gene encoding 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 facilitate protonation reduced glutathione) glutamine (H439Q). Both mutant genes were placed in same plasmid, pHD, where each them came under control a strong tac promoter. This designed achieve equal over-expression both E. cell. The parental homo-dimers show no or very little (H439Q) activity as reductases. formation vivo heterodimers, carrying one crippled fully functional active site, detected by absorbance spectroscopy fluorescence emission spectrometry FAD site complementation. fractional distribution homo- hetero-dimers accord that expected for random association enzyme subunits. In homo-dimer, H439Q mutation leads big fall value K$_{\text{m}}$ NADPH which binds some 1.8 nm from point (Berry, A., Scrutton, N. S. & Perham, R. Biochemistry 28, 1264-1269 (1989)). However, H439Q/C47S hetero-dimer exhibited kinetic parameters similar those wild-type enzyme. Thus, effect must be retained within accommodates it is not transmitted through protein second across subunit interface. ability generate reductase creates ideal system study protein--protein interactions molecular recognition at interface