In enzymology, catalytic reactions usually are investigated in vitro under well-defined conditions which may mimic physiological conditions. This experimental design allows the characterization of the kinetic capabilities of enzymes and to draw conclusions on both the mechanisms of the conversion of substrates into products and the effect of co-enzymes, essential metal ions and modifiers on the structure of the enzyme and thus on the overall catalytic reaction. In addition to biochemical methods, optical methods have emerged to study protein-structure function. In particular, cryogenic electron microscopy (cryo-EM) has led to new insights of the structures of large protein complexes at near-atomic resolution whose 3D models reveal how these molecules function in the cell.
However, the single-enzyme kinetics obtained under in vitro conditions may be misleading if this data is transferred into in vivo conditions due to the high viscosity in the cell. The cellular environment is densely packed with macromolecules such as proteins, RNA, DNA and metabolites which form ‘quinary’ interactions mediated by repulsing and attracting electrostatic forces. In particular, for metabolic pathways in which multi-step reactions are catalyzed this requires either a close spatial arrangement of the enzymes involved (and which has been proposed a substrate channeling for e.g. glycolysis) or lowered turnover rate through the pathway due to increased diffusion times for the individual substrates. Thus, the spatial and temporal arrangement of enzymes has to be regulated to achieve the necessary pathway efficiency, especially for those being located in the cytoplasm. In addition, pathway kinetics is highly controlled by modifying effectors e.g. the diverse types of inhibition and activation of individual enzymes.