Date of Award

4-19-2010

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor

Kathryn A. Thomasson

Abstract

Functional protein-protein interactions are essential for many physiological processes. For example, the association of glycolytic enzymes to F-actin is proposed to be one mechanism through which glycolytic enzymes are compartmentalized, and as a result, plays essential roles such as regulation of the glycolytic pathway, substrate channeling and increasing glycolytic flux. An accurate molecular picture describing such interaction, however, is not yet generalized. Similarly, questions about the specificity and extensive nature of such functional complexes in a variety of cells, tissues, or organisms, still remain ambiguous. Examination of these interactions in various animal species tests this hypothesis by observing whether binding sites are conserved across species. Specifically, Brownian dynamics (BD) simulations determine the energetics of the nonspecific and specific association of F-actin with three glycolytic enzymes: lactate dehydrogenase (LDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and fructose-1,6-bisphosphate aldolase (aldolase), from different species, as a function of ionic strength. Brownian dynamics (BD) also elucidates the interactions between the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and lactate dehydrogenase (LDH); and tests the hypothesis of whether the interaction between GAPDH and LDH produces a functional complex that can efficiently and reversibly transfer the cofactor NAD(H) between both enzymes. All the BD simulations are steered by electrostatics calculated by Poisson-Boltzmann theory. The electrostatic potentials about the protein models are obtained by numerically solving the Poisson-Boltzmann (PB) equation using both the full (FPB) and linearized (LPB) expressions. The BD results confirm experimental observations that the degree of association diminishes as ionic strength increases, but also suggest that these interactions are still significant at physiological ionic strength (0.1–0.15 M) and for some enzymes specific in nature. The Boltzmann population of glycolytic enzyme conformational states around F-actin determines the recognition patches responsible for interactions. These complexes are mainly stabilized by the enzyme's positively charged lysine and arginine residues and negatively charged glutamates and aspartates on the F-actin subdomain 1 binding surface. Stable complexes are formed between GAPDH and LDH. These complexes are mainly stabilized by positively charged lysine residues and negatively charged glutamates and aspartates from both GAPDH and LDH. The average transfer time of NAD from solution to free enzymes is 500 ns as compared to 56–320 ns when NAD is transferred between the active sites of a GAPDH/LDH complex. Channeling transfer is more efficient than solution transfer, due to active site proximity, favorable electrostatics and complex geometry.

Download

Share

COinS