Abstract
Glyceraldehyde 3-phosphate dehydrogenase and phosphoribulokinase exist as stable enzymes and as part of a complex in Chlamydomonas reinhardtii. We show here that phosphoribulokinase exerts an imprinting on glyceraldehyde 3-phosphate dehydrogenase, which affects its catalysis by decreasing the energy barrier of the reactions with NADH or NADPH by 3.8 +/- 0.5 and 1.3 +/- 0.3 kJ.mol(-1). Phosphoribulokinase and glyceraldehyde 3-phosphate dehydrogenase within the complex are regulated by NADP(H) but not by NAD(H). The activities of the metastable phosphoribulokinase and glyceraldehyde 3-phosphate dehydrogenase released from the complex preincubated with NADP(H) are different from those of the metastable enzymes released from the untreated complex. NADP(H) increases phosphoribulokinase and NADPH-glyceraldehyde 3-phosphate dehydrogenase activities with a (~)K(0.5 (NADP)) of 0.68 +/- 0.16 mm and a (~)K(0.5 (NADPH)) of 2.93 +/- 0.87 mm and decreases NADH-dependent activity. 1 mm NADP increases the energy barrier of the NADH-glyceraldehyde 3-phosphate dehydrogenase-dependent reaction by 1.8 +/- 0.2 kJ.mol(-1) and decreases that of the reactions catalyzed by phosphoribulokinase and NADPH-glyceraldehyde 3-phosphate dehydrogenase by 3 +/- 0.2 and 1.2 +/- 0.3 kJ.mol(-1), respectively. These cofactors have no effect on the independent stable enzymes. Therefore, protein-protein interactions may give rise to new regulatory properties.
Highlights
Enzymes do not exist as separate, independent entities in the cell but interact with many components, including membranes and other proteins, to form more complex structures
We show here that phosphoribulokinase exerts an imprinting on glyceraldehyde 3-phosphate dehydrogenase, which affects its catalysis by decreasing the energy barrier of the reactions with NADH or NADPH by 3.8 ؎ 0.5 and 1.3 ؎ 0.3 kJ1⁄7mol؊1
We conclude that protein-protein interactions are responsible for decreasing the energy barrier of the reactions catalyzed by the metastable glyceraldehyde 3-phosphate dehydrogenase (GAPDH) when NADH-dependent activity (Ϫ3.8 Ϯ 0.5 kJ1⁄7molϪ1) or NADPH-dependent activity (Ϫ1.3 Ϯ 0.3 kJ1⁄7molϪ1) is monitored
Summary
Enzymes do not exist as separate, independent entities in the cell but interact with many components, including membranes and other proteins, to form more complex structures. We have purified independent, stable, phosphoribulokinase (PRK) [8] and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), which are not included in the complex. The dissociated or metastable enzymes produced by dissociating a multi-enzyme complex by dilution may retain for a while some features of the conformation they had in the associated form One may wonder (i) if the GAPDH inserted in the complex we have purified could be regulated by these metabolites and (ii) if they alter the imprinting effects between these two enzymes This report examines these questions using kinetic and thermodynamic analyses
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