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Enzymes are biocatalysts that can increase the rate of chemical reactions with autonomous factors. While chemical reactions in water are often too slow to support life, the action of enzymes will increase the rate constant such that biological life becomes possible. The main factor that explains enzymatic rate enhancements is a lowering of the free energy of the transition state compound, and this is accomplished through for instance, tight binding to the compound, and activation of functional groups. To take the research-field forward is important to deeply understand how an enzyme catalyzes a biochemical reaction, robust enzyme production protocols must be developed together with kinetic studies and determination of three-dimensional structures. Here I have employed different techniques used for recombinant protein production, characterization of biophysical properties and catalytic parameters, and three-dimensional structure determination in order to expand the understanding of fundamental aspects of enzymology. Two different models of protein enzymes, Bombyx mori cocoonase and Escherichia coli adenylate kinase, were selected, which are categorized as protease and transferase. In paper I, we have successfully developed a stable and reproducible method for producing large amounts of functional recombinant Bombyx mori cocoonase by using an E. coli-based system which is beneficial over yeast and insect cell expression systems. To obtain a starting point for further structural studies, in paper II we have located serine 181 in Bombyx mori cocoonase as the catalytic nucleophile, making it highly suitable for the design of a stable serine variant. In order to define conditions where the enzyme is stable and suitable for structure determinations we have screened for suitable inhibitors, and we found that benzamidine hydrochloride is an effective inhibitor. In paper III, we provide a detailed picture of how Mg²⁺ ions activate the reversible phosphate transfer reaction catalyzed by adenylate kinase. Here, Mg²⁺ ions activate the positioning of substrates to achieve an optimal reaction angle that is critical for the chemical reaction. We also discovered a network of interactions involving amino acids and water molecules that are required for the correct positioning of Mg²⁺ ions. Using heavy water (deuterium oxide) as an alternative solvent, we discovered in paper IV that water molecules play an important role in enzymatic catalysis, structural stability, and coordination of indirect contacts with substrates or ligands of adenylate kinase. This implies that water plays indirect roles in reversible phosphoryl transfer. In paper V, we have worked to understand the chemical relationship between structural helices (terminal α-helix bending, fraying or unfolding, and order/folding in bacterial, archaeal, and human adenylate kinases, respectively) that are linked to large-scale conformational changes. We discovered that the flexibility of the α-helices' terminals can regulate the enzymatic dynamics and catalysis of adenylate kinase. In summary, our results contribute to the understanding of protein dynamics, structural flexibility, and changes linked to the catalytic function of enzymes.

Proteins are the workhorses of the cell and support all cellular functions governing biological life. The diversity of protein function is intimately linked to the unique threedimensional structure and intrinsic dynamics of each individual protein. One class of proteins, kinases, are enzymes that catalyze the transfer of a phosphoryl group from ATP to substrate molecules, thereby playing pivotal roles in cellular processes. There are different types of kinases acting on substrates ranging from a simple nucleotide to an entire protein. Aurora B  is a protein kinase with essential roles in cell division and is regulated by multiple mechanisms that ensure faithful mitotic progression. Adenylate kinase (AK) is a small nucleotide kinase that maintains the energy homeostasis in the cell by interconverting ATP and AMP into two ADP molecules.  In this thesis, the link between function, dynamics and structure has been explored in the two kinases Aurora B and AK by combining a selection of structural and functional studies. 

Studies on Aurora kinase B were focused on overcoming production limitations and exploring the effects of phosphorylation on protein dynamics and function. In Paper I, we developed a new production approach of human Aurora B in complex with its regulatory partner inner centromere protein (INCENP) in order to address previous production limitations. The developed approach was based on a fusion protein design with robust expression levels in E. coli, resulting in pure and fully functional protein with yields on the mg-scale. This approach enabled further structural and functional studies and was used to study the effects of phosphorylation on dynamics and substrate binding in Paper II. In this latter paper, we conducted a comparative study between the phosphorylated and dephosphorylated Aurora B:INCENP complex by probing protein dynamics with ¹⁹F NMR spectroscopy experiments. We found that the complex exists in a conformational equilibrium between inactive and active structural states, which is greatly influenced by phosphorylation and substrate binding.

The studies of AK aimed at understanding how small structural elements, such as termini of α-helices and protein bound water molecules, possibly facilitate catalysis. In Paper III, we investigated whether the termini of α-helices could support the large-scale conformational changes underlying catalysis in AK. We employed a comparative approach focused on a bacterial, archaeal and human AK, and found through a combination of X-ray crystallography, NMR spectroscopy and enzymatic activity assay experiments, that the inherent flexibility in the termini of α-helices influences the conformational changes correlated to catalysis. In Paper IV, the role of water molecules in AK catalysis was investigated by swapping the solvent from water to deuterium oxide and measure the effects on catalysis, substrate binding and protein stability with NMR spectroscopy, ITC and CD spectroscopy experiments. We showed that water molecules have an impact on AK catalysis, structural stability and coordination of indirect substrate contacts, all combined suggesting an indirect but important role of water molecules in the phosphoryl transfer reaction. Overall, this thesis contributes to an advanced understanding of how protein structure and dynamics modulate the catalytic function of kinases.