Abstract
Biological systems are highly regulated. They are also highly resistant to sudden perturbations enabling them to maintain the dynamic equilibrium essential to sustain life. This robustness is conferred by regulatory mechanisms that influence the activity of enzymes/proteins within their cellular context to adapt to changing environmental conditions. However, the initial rules governing the study of enzyme kinetics were mostly tested and implemented for cytosolic enzyme systems that were easy to isolate and/or recombinantly express. Moreover, these enzymes lacked complex regulatory modalities. Now, with academic labs and pharmaceutical companies turning their attention to more-complex systems (for instance, multiprotein complexes, oligomeric assemblies, membrane proteins and post-translationally modified proteins), the initial axioms defined by Michaelis-Menten (MM) kinetics are rendered inadequate, and the development of a new kind of kinetic analysis to study these systems is required. This review strives to present an overview of enzyme kinetic mechanisms that are atypical and, oftentimes, do not conform to the classical MM kinetics. Further, it presents initial ideas on the design and analysis of experiments in early drug-discovery for such systems, to enable effective screening and characterisation of small-molecule inhibitors with desirable physiological outcomes.
Highlights
Mechanistic enzymology plays a pivotal role in rational drug discovery efforts by providing critical insights into the nature of the target enzyme that is inhibited
Cooperative enzymes and approaches in drug discovery: Once an allosteric or cooperative modulator is identified, interpreting their mechanism of action can oftentimes be complicated by behaviours that do not conform to MM kinetics[76]
Covalent inhibitors are broadly classified as: (1) affinity labels that modify a functional group based on their activity or reactivity (2) quiescent affinity labels, which are similar to affinity labels but bind and modify the enzyme in a two-step process and use off-pathway mechanism for activation or (3) mechanism-based inhibitors, which are modified by the enzyme using its catalytic mechanism to produce a reactive doi:10.20944/preprints202010.0179.v1
Summary
Biological systems have evolved to maintain network robustness to overcome random disruptions of metabolic gradients[48]. Recent literature suggests that under excess receptor concentration and high affinity of the first ligand binding event (K1), negative cooperativity can function as a ligand sink depleting the ligand. This can result in binary sensitivities to changes in ligand concentration by filtering out small stimuli while acting sharply at high concentration of the ligand beyond a threshold. This can lead to complex system-level behavior with decisive bimodal switches that can give rise to bistability and oscillations[75]
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