Date of Award


Document Type


Degree Name

Master of Science (MS)


College of Science and Mathematics


Chemistry and Biochemistry

Thesis Sponsor/Dissertation Chair/Project Chair

John J. Siekeirka

Committee Member

Nina M. Goodey

Committee Member

Elena Petroff


The disease, Leishmaniasis, is caused by the protozoal parasite Leishmania, which is transmitted by the bite of an infected Phlebotomine female sandfly. It is a significant health problem in tropical regions of the world and new therapeutic approaches for treating this disease are urgently needed.

Protozoal MAPKs (Mitogen Activated Protein Kinases) play important roles in parasite viability and infectivity and as such, represent viable drug targets. It has been demonstrated that LmxMPKl is an essential MAPK required for the parasite to establish infection and for proliferation of the amastigote stage (the mammalian stage of the parasite) of the parasite. In our study we focused on LmxMPKl (Leishmania mexicana mitogen-activated protein kinase 1), which shares sequence homology to human ERK8 and p38 MAPK.

In our study we have successfully expressed LmxMPKl enzyme in HEK293T cells and BL21-AI cells. Mammalian expressed LmxMPKl was largely inactive but demonstrated low level basal activity as assessed by incorporation of the [y p] labeled ATP into MBP. In this assay activity was inhibited by BIRB796. On the other hand, bacterial expressed LmxMPKl, when assayed using an assay measuring consumption of ATP, was active but did not phosphorylate MBP which suggests we are measuring autophosphorylation of the enzyme itself. Interestingly, BIRB796 did not exhibit inhibitory activity in this assay.

In collaboration with Dr. Goodey, the direct binding of BIRB796 to active and inactive (dephosphorylated) LmxMPKl was assessed using tryptophan fluorescence quenching. Surprisingly, BIRB796 exhibited high affinity binding only to active LmxMPKl.

My results suggest that BIRB796 binds to active LmxMPKl with high affinity, but does not result in inhibition of the enzyme. This unusual result may indicate varying conformational or phosphorylation states of the enzyme depending upon the source (i.e. mammalian or bacterial) of recombinant enzyme.

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