Date of Award

12-2007

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Legacy Department

Biochemistry

Advisor

Morris, James C

Committee Member

Cao , Weiguo

Committee Member

Moore , Brandon

Committee Member

Powell , Gary

Committee Member

Temesvari , Lesly

Abstract

Trypanosoma brucei expresses two hexokinases (TbHK1 and TbHK2) that are 98% identical at the amino acid level. We previously found that recombinant TbHK1 (rTbHK1) has hexokinase activity, while rTbHK2 did not, a finding attributed to differences in the C-termini of the proteins. We found that the C-terminal tails of TbHK contained a novel motif specific for eukaryotic hexokinases found within the final helix of an ATP-binding domain. By altering the residues in the C-terminal tail of TbHK1 to resemble the residues found in TbHK2, we found that D454, F462, M466, and N469 were essential to TbHK1 catalysis. Additionally, we found that TbHK1 residues I458 and V468 contributed to substrate specificity as variants at these residues had a decreased affinity for fructose.
Here, we demonstrate that TbHK1 is essential to the bloodstream form of the parasite as RNA interference knockdown of the enzyme was lethal to the cell. Since TbHK1 is essential to the parasite, TbHK1 can be used as a target for drug development. Lonidamine, an inhibitor of tumor hexokinase, was shown to be an inhibitor of TbHK1 and hexokinase activity in whole cell lysates with IC50 of 0.850 mM and 0.965 mM, respectively. Moreover, we found lonidamine was toxic to bloodstream form parasites (LD50 = 0.050 mM) and insect (procyclic) form parasites (LD50 = 0.180 mM). Overexpression of TbHK1 in the procyclic parasites provided protection from lonidamine treatment.
We have also identified myristate as an uncompetitive inhibitor of TbHK1 (Ki = 0.050 mM) that also disassembles the hexameric protein complex of TbHK into monomers. Recombinant TbHK1 and TbHK2 can reassemble into hexamers upon removal of myristate, and oligomerization of TbHK1 with TbHK2 causes an increase in the hexokinase activity when compared to TbHK1 homohexamers. Surprisingly, assembly of TbHK2 with an inactive variant of TbHK1, S160A, resulted in an active complex. Given TbHK1:S160A is incapable of catalysis, our results indicate that TbHK2 is competent for hexokinase activity. Our studies have helped to improve our understanding of the composition and biochemistry of TbHK within T.brucei.

Included in

Biochemistry Commons

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