Alternative proton donors/acceptors in the catalytic mechanism of the glutathione reductase of Escherichia coli: the role of histidine-439 and tyrosine-99

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Abstract
The cloned Escherichia coli gor gene encoding the flavoprotein glutathione reductase was placed under the control of the tact promoter in the plasmid pKK233-3, 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 mutant retain .apprx. 1% and .apprx. 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 .apprx. 1% of the wild-type specific activity. Thus, Tyr-99 is not acting as a surrogate proton donor/acceptor to confer activity on the H439Q mutant, and it is unlikely that the phenolic hydroxyl group plays any role in proton transfer in the wild-type enzyme. We conclude that the imidazole side chain of His-439 probably acts as proton donor/acceptor in the wild-type enzyme, leading to an improvement in the kcat of approximately 100-fold compared with the H439Q and H439A mutants. The mutants may be functioning by recruiting another as yet unidentified protein side chain to act in this capacity, but it could be that the proton is simply acquired from solution, give the somewhat open structure of this part of the active site. The H439Q, H439A, and Y99FH439Q mutants all displayed a much lowered Km for NADPH compared with the wild-type enzyme, although NADPH is bound in a separate site some distance ( .apprx. 18 .ANG.) from that responsible for binding glutathione. The structural basis for this effect remains to be determined.

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