Engineering surface charge. 1. A method for detecting subunit exchange in Escherichia coli glutathione reductase.
The gene gor encoding Escherichia coli glutathione reductase was mutated to create a positively charged N-terminal extension consisting of five arginine residues followed by a factor Xa cleavage site to the enzyme polypeptide chain. The modified protein assembled in vivo to yield a dimeric enzyme with kinetic parameters indistinguishable from those of wild-type glutathione reductase. The N-terminal extension could not be released by treatment with factor Xa but could be removed by exposure to trypsin, again without effect on the enzyme activity. The modified enzyme was readily separated from the wild-type enzyme by means of ion-exchange chromatography or nondenaturing polyacrylamide gel electrophoresis. Incubation of the modified and wild-type enzymes, separately or as a mixture, with NADH led to their partial inactivation, and activity was restored by exposure to 1 mM reduced glutathione. No hybrid dimer was formed in the mixture of modified and wild-type enzymes, as judged by polyacrylamide gel electrophoresis, strongly suggesting that the inactivation induced by NADH was not due to dissociation of the parental dimers. The addition of otherwise benign positively or negatively charged extensions to the N- or C-terminal regions of the constituent polypeptide chains of oligomeric enzymes offers a simple route to detecting hybrid formation and the causative subunit dissociation and exchange.
Deonarain
M P
MP
Department of Biochemistry, University of Cambridge, U.K.
Scrutton
N S
NS
Perham
R N
RN
D000595:Amino Acid Sequence; D001483:Base Sequence; D010450:Endopeptidases; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005980:Glutathione Reductase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013499:Surface Properties
D011994:Recombinant Proteins; D005980:Glutathione Reductase; D010450:Endopeptidases