Substrate Channelingviaa Transient Protein-Protein Complex: The case of D-Glyceraldehyde-3-Phosphate Dehydrogenase and L-Lactate Dehydrogenase

Abstract

Background: D-Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and L-lactate dehydrogenase (LDH) can form a complex that can regulate the major metabolic pathways, however, the exact mechanism remains unknown. We analyzed a possibility of NADHchanneling from GAPDH-NADH complex to LDH isozymes using enzymes from different cells. Results: Enzyme-kinetics and NADH-binding studies showed that LDH can use GAPDH-NADH complex as a substrate. LDH activity with GAPDH-NADH complex was challenged with anti-LDH antibodies to show that the channeled and the diffusive reactions always take place in parallel. The channeling path is dominant only in assays with limiting free-NADH concertation that mimic cytosolic conditions. Analytical ultracentrifugation showed that the channeling does not require a high affinity complex. Molecular dynamics calculations and protein-protein interaction studies showed that LDH and GAPDH can form a leaky channeling complex only at subsaturating NADH concentrations. The interaction sites are conserved between LDH isozymes from heart and muscle, and between GAPDH molecules from rabbit and yeast cells. Positive electric fields between the NAD(H) binding sites on LDH and GAPDH tetramers, showed that NAD(H)-channeling within the LDH-GAPDH complex, can be an extension of NAD(H)channeling between the adjacent subunits in each tetramer. Conclusions: In the case of a transient (GAPDH-NADH)-LDH complex, the relative contribution from the channeled and the diffusive paths depends on the overlap between off-rates for the transient (GAPDH-NADH)-LDH complex and off-rates for the GAPDH-NADH complex. Molecular evolution or metabolic engineering protocols can exploit substrate channeling for metabolic flux control by fine-tuning substrate-binding affinity for the key enzymes in the competing reaction paths. Highlights-Substrate channeling molecular mechanism can regulate energy production and aerobic and anaerobic metabolism in cells -LDH and GAPDH can form a channeling complex only at sub-saturating NADH concentration -Channeled and diffusive paths always compete and take place in parallel -NADH channeling does not require a high-affinity complex -NADH channeling within GAPDH-LDH complex is an extension of NAD(H) channeling within each tetramer -Allosteric modulation of NADH binding affinity in GAPDH tetramer can regulate NAD(H) channeling Introduction D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and L-lactate dehydrogenases (LDH) are two NAD(H) dependent dehydrogenases that participate in glycolytic and gluconeogenic pathways 1,2 . Glycolysis and gluconeogenesis are the two major metabolic pathways that provide cells with metabolic precursors and a rapid source of energy. Parallel to glycolysis GAPDH participates in microtubule bundling, DNA replication and repair, apoptosis, the export of nuclear RNA, membrane fusion, and phosphotransferase activity 1,2 . GAPDH is implicated in Huntington's disease 3 , prostate cancer, and viral pathogenesis 1,2 . GAPDH could be a target of nitric oxide 4 and a target of ...

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