The C-terminal extension of glyceraldehyde-3-phosphate dehydrogenase subunit B acts as an autoinhibitory domain regulated by thioredoxins and nicotinamide adenine dinucleotide.
The regulatory isoform of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a light-activated enzyme constituted by subunits GapA and GapB. 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 redox dependence of recombinant GapB (E(m,7.9), -347 +/- 9 mV) was similar to native GAPDH, whereas GapA was essentially redox-insensitive. GapB mutants having one or two C-terminal cysteines mutated into serines (C358S, C349S, C349S/C358S) were less redox-sensitive than GapB. Different mutants with other cysteines substituted by serines (C18S, C274S, C285S) still showed strong redox regulation. 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. 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. 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. This covalent modification is required for the NAD-dependent association into higher oligomers and inhibition of the NADPH-activity. By leading to GAPDH autoinhibition, thioredoxin and NAD may thus concur to the dark inactivation of the enzyme in vivo.
Sparla
Francesca
F
Laboratory of Plant Physiology, Department of Biology, University of Bologna, Via Irnerio 42, Bologna I-40126, Italy.
Pupillo
Paolo
P
Trost
Paolo
P
D000595:Amino Acid Sequence; D002736:Chloroplasts / Q000201:enzymology; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D018724:Spinacia oleracea / Q000201:enzymology; D013879:Thioredoxins / Q000378:metabolism*
D007527:Isoenzymes; D010940:Plant Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D009243:NAD; D013879:Thioredoxins; D009249:NADP; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases