Enzyme kinetics characterizes the catalytic behavior of enzymes, specifically focusing on reaction rates.
Catalysis is the process of accelerating a chemical reaction. As biological catalysts, enzymes speed up the rate of reaction but do not affect the equilibrium (Keq) or the (thermodynamically) favorable direction of the reaction.
A favorable (spontaneous) reaction is one in which the free energy of the products is lower than the free energy of the reactants (ΔG < 0). However, a thermodynamically favorable reaction may not proceed (at a perceptible rate) on account of a kinetic barrier, e.g., activation energy, which is where enzymes come in.
In general, reaction rate is directly proportional to the frequency of effective collisions between reactant molecules (collision theory). Higher reactant concentrations have a higher probability of collision. Similarly, in an enzyme-catalyzed reaction, an increase in the relative concentration of substrate will increase the reaction rate up to a maximum rate (with enzyme concentration held constant).
At the point where an enzyme is catalyzing reactions as fast as it can (maximum turnover), adding more substrate will not make any difference and the reaction rate is at its maximum, Vmax. Adding more enzyme at this point will allow reaction rate to continue to increase and define a new Vmax. (That is, Vmax is defined for a specific enzyme concentration.)
The Michaelis-Menten equation calculates the rate of reaction (v) using Vmax, the substrate concentration ([S]), and the Michaelis constant (Km). Km equals the substrate concentration required for the reaction rate to reach ½Vmax. As a constant, Km does not fluctuate with changes in enzyme concentration and is indicative of enzyme-substrate affinity. Enzyme-catalyzed reactions with high enzyme-substrate affinity will reach the ½Vmax benchmark at a lower substrate concentration (have a lower Km), whereas lower enzyme-substrate affinities will result in needing a higher substrate concentration to reach ½Vmax (have a higher Km).
An exception to the Michaelis-Menten equation are enzymes with multiple binding sites (often over multiple subunits) that undergo cooperativity, a case in which the binding of one ligand will increase the affinity for binding another ligand at a different site. Binding sites that are not substrate active sites are called allosteric sites, and enzymes that undergo a change in catalytic activity on account of a molecule binding at an allosteric site are referred to as allosteric enzymes.
Feedback regulation of an enzyme occurs when a product of the reaction binds to an allosteric site on the enzyme, affecting its catalytic activity. This effect can be positive, producing a change that increases enzyme-substrate affinity, or inhibitory, reducing the activity at the active site or inactivating it completely. Binding molecules in feedback regulation may also extend to other reactants and products in an enzyme's metabolic pathway, producing upstream or downstream effects.
The catalytic relationship between an enzyme and its substrate is sensitive to disruption from other binding molecules or conformational changes in the enzyme.
A competitive inhibitor is a molecule that is similar enough to an enzyme's substrate that it can compete for the space occupying the active site. While a competitive inhibitor is bound to the active site, substrate cannot be processed.
A non-competitive inhibitor is a molecule that binds to an allosteric site on the enzyme, causing a conformational change that decreases catalytic activity at the active site regardless of whether a substrate is already bound.
A mixed inhibitor is a molecule that binds to an allosteric site on the enzyme, causing a conformational change that decreases catalytic activity at the active site. Mixed inhibitors generally have a preference towards binding either the enzyme-substrate complex or the enzyme alone.
An uncompetitive inhibitor is a molecule that binds only to the enzyme-substrate complex, rendering it catalytically inactive.
|Inhibition type||Binding state||Binding site||Blocks substrate||Effect on Km||Effect on Vmax||Overcome by ↑[S]|
|Competitive||E||active site||yes||increases||no effect||yes|
|Noncompetitive||E or ES||allosteric site||no||no effect||decrease||no|
|Mixed||E or ES||allosteric site||no||increases or decreases||decrease||partially|
Enzymes often work in concert, forming biochemical pathways that use sequences of enzyme-catalyzed reactions to achieve an overall goal (e.g. glycolysis). Enzymes along a pathway that are specifically targeted for regulation of the pathway are referred to as regulatory enzymes.
The catalytic activity of an allosteric enzyme is regulated by a effector molecule (acting as an activator or inhibitor) that binds an allosteric site, resulting in a conformational change to the enzyme that either activates or inhibits the active site on the enzyme. In homotropic allosteric regulation the effector molecule is also the enzyme's substrate, while the effector in heterotropic allosteric regulation is not a substrate of the enzyme.
Covalent modification can either activate or deactivate an enzyme through the addition or removal of a modifier using a reversible covalent bond (e.g. phosphorylation).
A zymogen (or proenzyme; generally indicated by the suffix -ogen) is an inactive precursor that will undergo irreversible conversion to the final active form of an enzyme. Activation triggers include proteolytic cleavage of an activation segment, change in environmental pH, or cofactors.