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