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Enzymatic Redox Chemistry 139
FAD
NADPH NADP +
S S S S
FADH 2
GSH
G
S
S − S
FAD
GSH
FAD
G
S S
SH S −
G
Figure 6.24 Mechanism for glutathione reductase.
The mechanism of the glutathione reductase catalytic cycle is shown in
Figure 6.24. NADPH reduces the bound Xavin to FADH 2 , which in turn
reduces the active site disulphide into two reduced cysteine residues. Attack
of a free cysteine thiol onto the disulphide linkage of oxidised glutathione
generates one equivalent of reduced glutathione. Attack of the second free
cysteine thiol generates a second equivalent of reduced glutathione and regenerates
the active site disulphide.
A high-resolution X-ray crystal structure for the human glutathione reductase
has made possible extensive protein engineering studies on the glutathione
reductase active site. Close examination of the NADPH binding site reveals that
the 2 0 -phosphate of NADPH is bound by three positively charged residues: Arg-
218, His-219 and Arg-224 (see Figure 6.23b). Arg-218 and Arg-224 are strictly
conserved in the amino acid sequences of other Xavoprotein disulphide oxidoreductases
that use NADPH. However, the amino acid sequence of NADHspeciWc
lipoamide dehydrogenase contains methionine and proline, respectively,
at these positions, which are incapable of forming electrostatic interactions.
Mutation of either of these two arginine residues in the Escherichia coli
glutathione reductase enzyme (to methionine and leucine, respectively) gave
mutant enzymes whose k cat =K M values for NADPH were reduced by approximately
100-fold, whilst a mutant enzyme containing both mutations had a 500-
fold reduced k cat =K M . Mutation of four additional residues identiWed in the
NADPH binding site to the corresponding residues in the NADH-speciWc
lipoamide dehydrogenase gave a mutant enzyme with an eight-fold preference
for NADH over NADPH. The wild type enzyme in contrast has a 2000-fold
preference for NADPH over NADH. This type of study shows that enzyme
characteristics such as cofactor speciWcity can in principle be rationally modiWed
using protein engineering.
A closely related enzyme trypanothione reductase (TR) has been found in
Trypanosoma and Leishmania parasites which cause human diseases such as
sleeping sickness and Chagas’ disease. These parasites use a modiWed form of