Co-crystallization and enzyme kinetics of Mycobacterium tuberculosis
and Escherichia coli Ribose-5-phosphate-isomerase with
their substrate and new inhibiting substances
Eva Kowalinski
Tuberculosis is one of the most widespread diseases. About one third of the world population
carries the causing bacterium, Mycobacterium tuberculosis, and each year about two million
people die from this cruel illness. As bacteria that are resistant to known medications are
developing quickly, research aimed to find new drugs is needed.
The RAPID centre is a collaboration between several research groups in Uppsala aiming to
develop new drugs against the pathogens that cause tuberculosis and malaria. To be able to
undertake rational drug design, the exact structure of a drug target protein, most commonly an
enzyme of the organism, has to be determined. The structure is used as a basis for design of
chemical compounds that inhibit it. The compounds are tested on the enzyme and in positive
cases the structure of the inhibitors with their target protein is determined. This is to verify
binding and allow improvement of the compounds. Effective inhibitors may provide the leads
needed to develop actual drugs in pharmaceutical companies.
X-ray crystallography was the method used for determining the protein structure in this study.
Since proteins are too small for examination with a light microscope, protein crystals have to be
grown and X-rays have to be used for observation of their structures.
This work was focused on the enzyme ribose-5-phosphate isomerase, an enzyme that provides
building blocks for DNA and for several messenger molecules in the cell. It might be important
for survival of the organism. The structure of the enzyme had been determined before and some
inhibitors had been designed. In this study the structure of the protein with one known inhibitor
was determined and 44 new inhibitors were tested for inhibition of the enzyme. One of these
inhibited successfully.
To learn more about the biology of the enzyme, it was crystallized together with its natural
substrate and the complex structure was determined. This structure was very similar to ones
derived from crystallization together with a substrate-like substance, a fact that validates the
common use of substrate mimics for crystallization.
The structure of an inactive mutant of the same protein from another species, Escherichia coli,
was determined, but the hopes of seeing substrate, product or an interesting intermediate binding
to the protein were not fulfilled.
Degree project in Biology – Examensarbete i biologi 20 poäng
Biology Education Centre and Department of Cell and Molecular Biology, Uppsala University
Supervisors: Annette Roos, Professor Sherry Mowbray, Professor Torsten Unge