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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.