Alternative proton donors/acceptors in the catalytic mechanism of the glutathione reductase of Escherichia coli: the role of histidine-439 and tyrosine-99.
The cloned Escherichia coli gor gene encoding the flavoprotein glutathione reductase was placed under the control of the tac promoter in the plasmid pKK223-3, allowing expression of glutathione reductase at levels approximately 40,000 times those of untransformed cells. This greatly facilitated purification of the enzyme. By directed mutagenesis of the gor gene, His-439 was changed to glutamine (H439Q) and alanine (H439A). The tyrosine residue at position 99 was changed to phenylalanine (Y99F), and in another experiment, the H439Q and Y99F mutations were united to form the double mutant Y99FH439Q. His-439 is thought to act in the catalytic mechanism as a proton donor/acceptor in the glutathione-binding pocket. The H439Q and H439A mutants retain approximately 1% and approximately 0.3%, respectively, of the catalytic activity of the wild-type enzyme. This reinforces our previous finding [Berry et al. (1989) Biochemistry 28, 1264-1269] that direct protonation and deprotonation of the histidine residue are not essential for the reaction to occur. The retention of catalytic activity by the H439A mutant demonstrates further that a side chain capable of hydrogen bonding to a water molecule, which might then act as proton donor, also is not essential at this position. Tyr-99 is a further possible proton donor in the glutathione-binding pocket, but the Y99F mutant was essentially fully active, and the Y99FH439Q double mutant also retained approximately 1% of the wild-type specific activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Deonarain
M P
MP
Department of Biochemistry, University of Cambridge, U.K.
Berry
A
A
Scrutton
N S
NS
Perham
R N
RN
D001483:Base Sequence; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D005980:Glutathione Reductase / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011522:Protons; D014443:Tyrosine / Q000235:genetics*
D011522:Protons; D014443:Tyrosine; D006639:Histidine; D005980:Glutathione Reductase