Table 4.

Refined threshold concepts and knowledge statements

Name	Knowledge statement(s)	Biochemical ideas that are unlocked once this concept is understood	Connections that were invisible before deep understanding of the concept
Steady state	Living organisms constitute open systems, which constantly exchange matter and energy with their surroundings, yet net concentrations remain relatively constant over time. This dynamic, yet outwardly stable condition is referred to as a steady state.	Steady state is an emergent process that results from regulation of numerous biological reactions.	Once the condition of steady state is recognized, the purpose of complex regulatory systems in maintaining steady state and their connections to each other become apparent.
		Steady state is a metastable condition that can be maintained only because of constant input of energy from the environment.
			Once the metastable nature of steady state is recognized, the importance of multi-tiered energy storage systems (starch, glycogen, triglycerides, etc.) becomes apparent.
	“Steady” is not synonymous with chemically “stable.” Concentrations are determined by kinetic, rather than thermodynamic, factors. Hence, biological systems do not exist in a state of chemical equilibrium.
		Steady state defines the conditions of life under which chemical reactions take place in cells and organisms. Therefore an understanding of steady state is necessary in order to correctly contextualize all of biochemistry.
	If an organism reaches chemical equilibrium, its life ceases. Consequently, organisms have evolved extensive regulatory systems for maintaining steady-state conditions.
Biochemical pathway dynamics and regulation	Reactions and interactions in biological systems are dynamic and reversible.	Chemical drivers result in bulk (emergent) properties observed in biological systems.	Once these concepts are understood, predictions can be made about 1) how biochemical pathways are likely to respond to changes environmental conditions and 2) cause and effect of fluctuations in biochemical pathways.
	Directionality of processes depends on the free energy and relative concentrations of reactants and products available.	Enzyme-mediated regulatory mechanisms allow pathways to be sensitive and responsive to the needs of the organism.
	Observable flux is the net result of forward and reverse processes.	Enzymes act as gatekeepers rather than drivers of chemical change.
	Enzymes control rates of forward and reverse reactions.
	Enzyme activity is highly regulated.
The physical basis of interactions	Interactions occur because of the electrostatic properties of molecules. These properties can involve full, partial, and/or momentary charges.	Once this concept is understood, similarities between different types of interactions become clear. Although interactions are given different names, they are all based on the same electrostatic principles.	A core biochemical principle is that structure governs function. Correct understanding of noncovalent interactions is essential in integrating structure and function.
Thermodynamics of macromolecular structure formation	Interactions in biological systems almost always take place in aqueous solution.	Protein folding, the assembly of lipids into micelles and bilayers, the association of polypeptide subunits to form oligomeric proteins, base pairing of DNA and RNA molecules, and all other biological interactions are driven by a common set of thermodynamic forces.	When the entropic and enthalpic forces that drive processes like protein folding and binding are understood, predictions can be made about the conditions under which these events will occur and what effect perturbations, like mutations will have.
	Bulk interactions in an aqueous system have an entropic component.
	Enthalpic and entropic contributions are responsible for biological structure.
		The aqueous environment of the cell plays an active and essential role in biochemical structure formation.
Free energy	The tendency toward equilibrium drives biological processes.	Biological systems use favorable processes to drive less-favorable processes, which allows for maintenance of steady state.	Once this concept is understood, the relationship among free energy, equilibrium, and steady state becomes apparent.
	Differences in free energy drive the chemical transformations underlying biological function.
	By providing a direct link between a thermodynamically favorable reaction with a thermodynamically unfavorable one, enzymes enable biological systems to drive a normally unfavorable reaction by coupling it to one with a large and favorable free-energy change.
	Enzymes affect reaction rate, yet do not affect equilibrium position.