Inhibition of the thioredoxin-dependent activation of the NADP-malate dehydrogenase and cofactor specificity.
The chloroplastic NADP-malate dehydrogenase is activated by reduction of its N- and C-terminal disulfides by reduced thioredoxin. The activation is inhibited by NADP(+), the oxidized form of the cofactor. Previous studies suggested that the C-terminal disulfide was involved in this process. Recent structural data pointed toward a possible direct interaction between the C terminus of the oxidized enzyme and the cofactor. In the present study, the relationship between the cofactor specificity for catalysis and for inhibition of activation has been investigated by changing the cofactor specificity of the enzyme by substitution of selected residues of the cofactor-binding site. An NAD-specific thiol-regulated MDH was engineered. Its activation was inhibited by NAD(+) but no longer by NADP(+). These results demonstrate that the oxidized cofactor is bound at the same site as the reduced cofactor and support the idea of a direct interaction between the negatively charged C-terminal end of the enzyme and the positively charged nicotinamide ring of the cofactor, in agreement with the structural data. The structural requirements for cofactor specificity are modeled and discussed.
Schepens
I
I
Institut de Biotechnologie des Plantes, UMR 8618 CNRS, Université de Paris-Sud, Bâtiment 630, Orsay, France.
Johansson
K
K
Decottignies
P
P
Gillibert
M
M
Hirasawa
M
M
Knaff
D B
DB
Miginiac-Maslow
M
M
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002736:Chloroplasts / Q000201:enzymology*; D004220:Disulfides; D006801:Humans; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D050538:Malate Dehydrogenase (NADP+); D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*; D010084:Oxidation-Reduction; D010944:Plants / Q000201:enzymology*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013879:Thioredoxins / Q000378:metabolism*
D004220:Disulfides; D011994:Recombinant Proteins; D009243:NAD; D013879:Thioredoxins; D009249:NADP; D008291:Malate Dehydrogenase; D050538:Malate Dehydrogenase (NADP+)