The channeling hypothesis (i.e., that substrates or co-enzymes may move from one active site to another without equilibrating with the bulk medium) is still controversial in glycolysis because glycolytic enzymes are large and have very dynamic interactions that are hard to detect. Channeling has been demonstrated in the tricarboxylic acid cycle where enzyme-enzyme complexes have been crystallized and electrostatic channeling pathways clearly modeled. A variety of experimental methods have provided evidence that channeling may occur in glycolysis including sedimentation, co-pelleting, and electrophoresis. There is also evidence of a metabolon, a functional multienzyme complex, for the glycolytic pathway. Computer modeling simulations can demonstrate potential enzyme-enzyme complexes and follow the trajectories of ligands as they move from one active site to another in such complexes. Herein, exploration of the channeling hypothesis in glycolysis will begin by using Brownian dynamics (BD) computer simulations on the enzymes that involve the co-factor nicotine adenine dinucleotide (NAD). The channeling hypothesis will be tested not only on the substrates of these enzymes, but also on the NAD itself. BD can explore the potential for forming enzyme-enzyme complexes, the potential pathways of ligands, and the efficiency of the ligand binding when compared to isolated enzymes in solution. The exportation will involve several enzymes and different species to see if there is a general trend observed throughout nature, including common vertebrate systems, disease causing organisms, and microorganisms. The initial focus will be on the parts of the pathway involving NAD because the enzymes can be well modeled based on known crystal structures and because it may be possible to follow the NAD through fluorescence experiments. Example channeling simulations include: NAD between glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and lactate dehydrogenase (LDH), glyceraldehyde-3- phosphate (GAP) between fructose-1,6-bisphosphate aldolase (aldolase) and GAPDH, GAP between triose phosphate isomerase (TPI) and GAPDH, GAP among aldolase, TPI and GAPDH, and finally pyruvate between pyruvate kinase and LDH.

Public Health Relevance

The proposed work includes computer simulations useful not only for basic biomedical science (e.g, rabbit, human, rat, chicken, zebra fish, baker's yeast,) but also for infectious disease organisms (e.g., P. falciparum, trypanosomes, Leishmania, E. coli, M. tuberculosis) in an attempt to determine how ubiquitous the enzyme- enzyme interactions are and what kinds of channeling possibilities exist across species.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Academic Research Enhancement Awards (AREA) (R15)
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Macromolecular Structure and Function D Study Section (MSFD)
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Barski, Oleg
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University of North Dakota
Schools of Arts and Sciences
Grand Forks
United States
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