![]() By leading to GAPDH autoinhibition, thioredoxin and NAD may thus concur to the dark inactivation of the enzyme in vivo. This covalent modification is required for the NAD-dependent association into higher oligomers and inhibition of the NADPH-activity. We conclude that a regulatory disulfide, between Cys-349 and Cys-358 of the C-terminal extension of GapB, does form in the presence of oxidized thioredoxin. The C-terminal GapB mutants (C358S, C349S, C349S/C358S) were active tetramers unable to aggregate to higher oligomers in the presence of NAD, whereas other mutants (C18S, C274S, C285S) again behaved like GapB. ![]() ![]() Fully active GapB was a tetramer of B-subunits, and, when incubated with NAD, it associated to a high molecular weight oligomer showing low NADPH-dependent activity. Different mutants with other cysteines substituted by serines (C18S, C274S, C285S) still showed strong redox regulation. GapB mutants having one or two C-terminal cysteines mutated into serines (C358S, C349S, C349S/C358S) were less redox-sensitive than GapB. ![]() The redox dependence of recombinant GapB ( E m ,7.9, −347 ± 9 mV) was similar to native GAPDH, whereas GapA was essentially redox-insensitive. Phe-Arg-Phe(4NO3) by 23 nM of cathepsin X used to calculate catalytic. The NADPH-dependent activity of regulatory GAPDH from spinach chloroplasts was affected by the redox potential ( E m ,7.9, −353 ± 11 mV) through the action of thioredoxin f. The regulatory isoform of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a light-activated enzyme constituted by subunits GapA and GapB. ![]()
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