USE OF TRANSFERRED NUCLEAR-OVERHAUSER-EFFECT SPECTROSCOPY TO MEASURE THE BOUND CONFORMATION OF A DISULFIDE-REPLACED ANALOG OF GLUTATHIONE DISULFIDE AS AN INHIBITOR OF YEAST GLUTATHIONE-REDUCTASE
Kj. Embrey et al., USE OF TRANSFERRED NUCLEAR-OVERHAUSER-EFFECT SPECTROSCOPY TO MEASURE THE BOUND CONFORMATION OF A DISULFIDE-REPLACED ANALOG OF GLUTATHIONE DISULFIDE AS AN INHIBITOR OF YEAST GLUTATHIONE-REDUCTASE, European journal of biochemistry, 221(2), 1994, pp. 793-799
The analogue of glutathione disulphide (GSSG) in which the disulphide
bridge of GSSG is replaced by -CH2-S- was synthesised from L-cystathio
nine using t-butoxycarbonyl and t-butyl ester protection with triethyl
silane-promoted deprotection. This analogue (GCSG) was found to be a l
inear, competitive inhibitor of yeast glutathione reductase (K-i value
981 mu M at pH 7.0), a very poor substrate and not to act as an irrev
ersible inhibitor of glutathione reductase. The weak binding of GCSG t
o glutathione reductase permitted the use of transferred nuclear Overh
auser effect spectroscopy (TRNOESY) to investigate the bound conformat
ion of GCSG in its complex with glutathione reductase. The solution st
ructure of free GCSG was investigated by NMR spectroscopy using a rang
e of NMR techniques. The TRNOESY experiment allowed a range of conform
ations to be determined for the central bridge region (containing the
-CH2-S- replacement) of GCSG bound to yeast glutathione reductase. Usi
ng the nuclear Overhauser effect constraints thus derived, in combinat
ion with molecular graphics and energy minimisation based on the known
crystal coordinates of glutathione disulphide (GSSG) bound to human e
rythrocyte glutathione reductase, allowed an explanation of the lack o
f substrate activity of GCSG, its inactivity as a suicide inactivator
and its relatively weak binding in terms of the enforced mislocation o
f the -CH2-S- bridge with respect to the catalytic residues (relative
to GSSG). Thus, the simple replacement of -S- by -CH2-, common in medi
cinal chemistry, can lead to poor receptor binding if the replacement
occurs in a central, rather than peripheral, part of the ligand under
modification.