Probing the kinetic mechanism and coenzyme specificity of glutathione reductase from the cyanobacterium Anabaena PCC 7120 by redesign of the pyridine-nucleotide-binding site

Glutathione reductase from the cyanobacterium Anabaena PCC 7120 contains a pyridine-nucleotide-binding motif differing from that of the enzyme from ot her sources and an insertion of 10 amino acid residues. Homology modeling w as used to obtain a model of the enzyme structure. It revealed that in the Anabaena enzyme Lys(203) replaces Arg, found to interact with the 2'-phosph ate of NADP(H) in the enzyme from other sources, and that it has an extra l oop near the entrance of the pyridine-nucleotide-binding site. The steady-s tate and preequilibrium kinetic properties were characterized for the wild- type enzyme, a K203R, and a loop deletion mutant. All enzyme forms had high er catalytic efficiency with NADPH than with NADH, although the difference was less than for glutathione reductase from other sources. The specificity was most pronounced in the formation of the charge-transfer complex betwee n the pyridine nucleotide and oxidized enzyme-bound FAD, as compared to lat er steps in the reaction. Unexpectedly, by replacing Lys(203) with Arg, the specificity for NADPH was diminished in the complete redox reaction. Ser(1 74) appears to interact with the 2'-phosphate of NADPH and introduction of arginine instead of lysine, therefore, has Little effect on the interaction with this coenzyme. However, the efficiency in forming the charge-transfer complex between the pyridine nucleotide and oxidized enzyme-bound FAD was increased in the K203R mutant using NADPH but not with NADH. The lack of af finity toward 2',5'-ADP-Sepharose by the wild-type enzyme was not changed b y replacing Lys(203) with Arg but deletion of the loop resulted in an enzym e that bound to the immobilized ligand. Removal of the loop increased the e fficiency of the enzyme in the reductive half-reaction with both pyridine-n ucleotides as well as in the overall catalytic mechanism.