Engineering surface charge. 2. A method for purifying heterodimers of Escherichia coli glutathione reductase.
Two gor genes encoding different mutants of Escherichia coli glutathione reductase have been expressed in the same E. coli cell, leading to the creation of a hybrid form of the enzyme dimer. One of the gor genes carried, in addition to various directed mutations, a 5' extension that encodes a benign penta-arginine "arm" added to the N-terminus of the glutathione reductase polypeptide chain [Deonarain, M.P., Scrutton, N.S., & Perham, R.N. (1992) Biochemistry (preceding paper in this issue)]. This made possible, by means of ion-exchange chromatography or nondenaturing polyacrylamide gel electrophoresis, the facile separation of the hybrid enzyme from the two parental forms. Moreover, the two subunits in the hybrid enzyme could be made to carry different mutations. In this way, glutathione reductases with only one active site per dimer were generated: the effects of replacing tyrosine-177 with glycine in the NADPH-binding site, which greatly diminishes the Km for glutathione and switches the kinetic mechanism from ping-pong to ordered sequential, and of replacing His-439 with glutamine in the glutathione-binding site, which greatly diminishes the Km for NADPH, were both found to be restricted to the one active site carrying the mutations. This system of generating separable enzyme hybrids is generally applicable and should make it possible now to undertake a more systematic study of catalytic mechanism and assembly for the many enzymes with quaternary structure.
Deonarain
M P
MP
Department of Biochemistry, University of Cambridge, U.K.
Scrutton
N S
NS
Perham
R N
RN
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005980:Glutathione Reductase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013057:Spectrum Analysis; D013499:Surface Properties
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D005980:Glutathione Reductase