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Abstract

Enzymes are the catalysts of life. They accelerate the rate of chemical reactions that would otherwise take longer than an organism’s lifetime to take just millisecond. To achieve these remarkable rate enhancements enzymes arrange into a three dimensional fold that places its amino acids in a way, which binds the transition state of the reaction better than the substrates and products of the reaction, thereby lowering the activation energy of the reaction. Enzymes are also very specific and often only catalyze one specific chemical transformation without producing side products. They are able to achieve all this under ambient temperatures and in cells that contain over 2700 different metabolites. In this work we focus on the mechanisms enzyme use to control reactive intermediates both inside their active site and between enzymes of a metabolic pathway to avoid the formation of deleterious side products. In the first part we investigate the catalytic cycle of NAD(P)H dependent oxidoreductases. We show that the two enoyl-thioester reductases; Etr1p from Candida tropicalis of the MDR enzyme superfamily and InhA from Mycobacterium tuberculosis of the SDR enzyme superfamily form a covalent adduct between substrate and the C2 carbon of the cofactor. The observation of this reactive intermediate at the active site of enzymes from the two largest NAD(P)H dependent oxidoreductase superfamilies not only calls for a careful reconsideration of the canonical reaction mechanism of these enzymes, but also sets the basis for the development of novel tools to study, manipulate and inhibit their catalytic cycle. We demonstrate this by successfully changing the protonation specificity of Etr1p from re- to si- face. Using the molecular probe we show that a conserved threonine at the active site of Etr1p is mainly responsible for preventing the formation of a toxic side product and not for the stabilization of the wanted transition state along the reaction coordinate. This effect of destabilization of unwanted transition states, often termed ´negative catalysis´, poses a complementary mechanism of reaction control to the canonical transition state theory and is discussed in detail in this work. In the second part of this thesis we take a look at two enzyme complexes and the strategies they use to control the transfer of a reactive intermediate from one active site to the next one. The trifunctional propionyl-CoA synthase forms a closed reaction chamber to sequester the reactive acrylyl-CoA intermediate. This reaction chamber encloses all three active sites of the enzyme fusion protein, but does not show the directionality of a conventional tunnel, and the CoA ester intermediates are not covalently attached to the enzyme but freely diffuse within the compartment. The substrate channeling mechanism of the thiolase/HMG-CoA synthase complex of archaea most closely resembles the covalent swinging arm fatty acid and polyketide synthases use to channel their intermediates. In the thiolase/HMG-CoA synthase complex the intermediate is however not covalently attached, but instead tightly bound in a shared CoA binding site, enabling the pantothenyl-arm of CoA to swing from the thiolase active site to the HMG-CoA synthase active site. The two channeling systems we describe in this work therefore represent two alternative ways of channeling CoA ester intermediates in a non-covalent fashion.