Date of Award

2-5-2007

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor

Kathryn A. Thomasson

Abstract

The association of glycolytic enzymes with cytoskeletal proteins such as actin is thought to be a structural basis for compartmentation of the glycolytic pathway. It is also suggested that such interactions bring glycolytic enzymes within close proximity to each other so that substrate transfer between sequential enzymes is faster. Brownian dynamics (BD) simulations were used to study the binding of glyceraldehydes-3-phosphate dehydrogenase (GAPDH) to actin and fructose- 1,6-bisphosphate aldolase (aldolase) in different species, and the dynamics of substrate binding. High-resolution three-dimensional models of the proteins used in the simulations were either obtained from X-ray structures or built by homology modeling. The electrostatic potential about each protein for use in the BD simulations was predicted by solving the linearized Poisson-Boltzmann equation. The simulations revealed favorable interactions between GAPDH and F-actin that were electrostatic in nature. Analysis showed that positively charged patches on the GAPDH structure interacted with negatively charged residues in subdomain I of actin. These interactions were greatly diminished when these charged amino acid residues were neutralized, hence confirming the importance of the identified residues and emphasizing the electrostatic nature of these interactions. The BD simulations also showed that favorable interactions do occur between GAPDH and aldolase, producing complexes that could be useful for substrate channeling. Simulations of substrate binding dynamics revealed that the binding of glyceraldehydes-3-phosphate (GAP) to GAPDH involves electrostatic steering, which enhances the first encounter bimolecular rate. The efficiencies of transfer of GAP from solution to GAPDH were lower than efficiency of transfer from an aldolase active site in a complex. Also, the transfer times were much shorter, when GAP was originating from an aldolase active site. This suggests that there is a possibility of substrate channeling between aldolase and GAPDH. The enzyme/actin interactions were complemented and supported by cosedimentation experiments and an analysis of data from viscometric measurements. Results of these experiments showed that enzyme/actin interactions in muscle were of higher affinity than those in yeast and that the tetrameric enzymes were capable of crosslinking actin filaments.

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