Kinetic evidence for protein complexes between thioredoxin and NADP-malate dehydrogenase and presence of a thioredoxin binding site at the N-terminus of the enzyme.

The kinetics of activation of NADP-malate dehydrogenase (MDH; EC 1.1.1.82) from soybean and spinach leaves by the chloroplast thioredoxins isolated from the same plants, by the corresponding storage forms of the soybean chloroplast thioredoxins from soybean seeds, and by the bacterial Escherichia coli thioredoxin have been studied. The Hill equation has been applied to evaluate the saturation kinetics. The observed variable thioredoxin saturation characteristics (Vmax 0.37-14.5 mumol NADPH min-1 mg enzyme-1; K0.5 0.15-1.33 microM; Hill coefficient h 0.90-3.04) indicate that the activation of NADP-MDH depends strongly on the individual thioredoxin used. Thus, thioredoxin action is not solely due to simple reductive activation of the NADP-MDH. Specific thioredoxin complex formation between thioredoxin and NADP-MDH must be included into the mechanism of the activation process. To study the regulatory consequences of the specific thioredoxin/NADP-MDH complexes we investigated the saturation kinetics of the substrates NADPH and oxaloacetate in presence of different concentrations of each individual thioredoxin species. The kinetic characteristics of the substrates (S0.5, Vmax, and Hill coefficients h) varied individually in response to the different thioredoxin species substantiating our model of thioredoxin/ NADP-MDH complex formation. Aminopeptidase-K-truncated pea NADP-MDH has been used to demonstrate that the N-terminal 37 amino residues are involved in providing a specific thioredoxin binding site. The fact that the versatile light-dependent regulation of numerous enzyme activities by only two thioredoxin species in chloroplasts cannot be accomplished without the formation of thioredoxin/target enzyme complexes is discussed in detail.