Date of Award

8-11-2008

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor

Kathryn A. Thomasson

Abstract

One potentially important step in the compartmentation of glycolysis is the binding of glycoltic enzymes including: fructose-1 .6-biphosphate aldolase (aldolase), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or lactate dehydrogenase (LDH) isoforms to cytoskeletal proteins like: F-actin or tubulin which is the major building block of microtubules. Tetrameric LOH can have different isoforms resulting from all possible combination of the muscle (M) or heart (H) type monomers. The LDH mixed tetrameric isoforms, LDH-M3H, LDH-MMHH, LDH-MHMH, LDH-MHHM and LDH-MH3, give distinctive electrostatic potentials and charges. If the positive electrostatic potential region between subunits A/D and subunits B/C, remains intact (such as in M3H) the mixed isoforms bind F-actin. In rabbit mixed isoforms, the positive electrostatic groove is preserved when at least one muscle subunit is involved in forming the groove, whereas human mixed isoforms require two adjacent units be of the muscle type, to preserve the positive groove. This difference in the electrostatic picture results from differences in the primary structure of the human and rabbit heart LDH subunits, leading to a net charge of -2 e for a human heart subunit when compared to rabbit. Hence more rabbit mixed isoforms bind F-actin when compared to human mixed isoforms. Brownian dynamics provide first-encounter snapshots between three glycolytic enzymes with pig tubulin. The enzymes examined were brain aldolase, muscle aldolase, GAPDH and muscle LDH. Four models of tubulin which differed only in the orientation of some of their C-termini residues were obtained from molecular dynamics (MD) simulations. The negatively charged residues of the tubulin C-termini dominated interactions with positively charged regions of the enzymes, for muscle aldolase, GAPDH and LDH, although other regions of tubulin were also important. For brain aldolase, positively charged residues in the C-termini of tubulin interacted with negatively charged residues of aldolase. The strongest interactions resulted from tubulin models with the C-termini or α- and β-subunits pointing away from each other as predicted by the free energy profiles the for interactions. Complexes between aldolase and GAPDH with tubulin were predominantly due to interaction of one subunit of the enzyme with one tubulin subunit. The LDH-M4/tubulin interaction was typically characterized by association of two LDH subunits with one or two subunits of tubulin. The different MD models of tubulin only varied in the most important C-terminal residues involved in binding the enzymes. While the Maestro and the Original model bound more with the end C-terminal residues (E448 and E449), the CHARMm and the Discover models preferred to bind with earlier C-termini (E444, E445 and E446) residues. These results agree with experimental studies, which suggest that tubulin C-termini bind aldolase, GAPDH and LDH-M4. Enzyme interactions with cytoskeletal proteins suggest a platform for glycolytic enzyme recruitment, which may lead to substrate channeling and a more efficient glycolysis.

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