Topic 5E: enzymes

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6 classes of enzymes:

Oxidoreductases: catalyze redox reactions or reactions involving electron or H+ transfer

Transferase: catalyze reactions in which groups are transferred from 1 place to another

Hydrolases: regulate hydrolysis reactions

Lyases: catalyze the addition of functional groups to double bonds, or the formation of double bonds via removal of functional groups

Isomerases: catalyze reactions in which isomers are produced via group transfers WITHIN a molecule

Ligases: catalyze coupled condensation reactions

other important enzyme classes: kinases, phosphatases, and proteases

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Characteristics of enzymes:

Specificity: a single enzyme typically interacts only with a single class of substrates

Each enzyme has an optimum temperature and pH for activity; changes in conditions can lead to denaturation, which can be irreversible sometimes

have no effect on reaction equilibrium, no effect on thermodynamic spontaneity

Mechanism of action:

the enzyme-substrate complex lowers activation energy of the reaction, thereby speeding up reaction rate

work by bringing reactants in close proximity to one another (covalent catalysis, acid-base catalysis)

Induced fit model: substrate binds to active site, conformational change occurs to tighten binding b/w enzyme and substrate, to facilitate the reaction.

Ensures the correct substrate binds to the enzyme, as if the substrate does not have the right shape, conformational change does not happen.

Formerly, lock and key model was popular theory

cofactors: ions that bind to the enzyme that are required for enzyme to be able to function

coenzymes: organic materials typically derived from vitamins or diet that bind to enzyme and are essential for enzyme function

substrate binding mechanisms:

ping-pong mechanism: aka double-displacement reaction, change in enzyme into intermediate form when the first substrate is changed to product

ordered mechanism: 1 substrate must bind 1st before the next substrate can bind

random sequential mechanism: substrates and products are bound and released in random order

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Kinetics:

Michaelis-Menten mechanism: describes the relationship between initial rection rate and [substrate]

[substrate] and [enzyme] are directly related to rate of reaction/activity of enzyme

rate of rxn can be measured by the appearance of product or disappearance of substrate

as [substrate] increase, rate of reaction decreases.

Saturation: point beyond which [substrate] has no effect on reaction rate.

This is a fundamental characteristic of enzyme-catalyzed reactions and is NOT seen in uncatalyzed reactions.

The Michaelis constant (Km) is also an indicator of enzyme affinity; they are inversely related.

Vmax and Km are constants for any given enzymes; they are INDEPENDENT of [substrate]

Vmax is a function of [enzyme]; Vmax will increase if the reaction is run at a higher enzyme concentration

double-reciprocal plots can be used to analyze enzyme activity using Michaelis-Menten relationships

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Regulation of enzyme activity:

Proteolytic cleavage: aka irreversible covalent modification, the cleavage of specific bonds on the enzyme can activate or destroy a zymogen (proenzyme, the enzyme in its active form)

Reversible covalent modification: activation vs deactivation via phosphorylation or the addition of another modifier (like AMP); this is reversible because the covalent bond b/w the enzyme and the modifier can be hydrolyzed

Control proteins: protein subunits that associate with certain enzymes and modulate their activity (i.e. G proteins)

Allosteric interactions: the modification of an enzyme’s configuration via binding of an activator or inhibitor at the allosteric site

when a repressor molecule binds to the allosteric site, there is conformational change in active site, so substrate is not able to bind anymore, and enzyme activity is halted or decreased. This is a reversible process.

Activator molecules can also bind, and in some enzymes, they are only active when the activator is bound to the allosteric site

cooperativity: allosteric regulation observed in proteins made of multiple identical subunits (like Hgb)

the binding of substrate to 1 subunit increases the other unbound subunits’ affinity for substrate by inducting a conformational change

Hill coefficient: measure of cooperativity in a binding process; if >1, shows positive cooperativity binding, coefficient of 1 indicates independent binding

Inhibitors:

Competitive: similar in shape to substrate, competes with the substrate to bind to the active site, which ultimately decreases enzyme activity.

Increasing [substrate] does help increase enzyme activity

Noncompetitive (allosteric): bind to allosteric sites and change active site conformation to decrease enzyme activity. Substrate is still able to bind, but it is not catalyzed

Increasing [substrate] has ZERO impact on enzyme activity

Mixed inhibitor: bind at a site other than the active site, so they do not prevent the substrate from binding. Able to bind the enzyme itself, and/or the enzyme-substrate complex. They ALL lower Vmax.

Km will increase if the mixed inhibitor behaves like a competitive inhibitor (binds the enzyme before substrate)

Km will decrease if the mixed inhibitor binds the enzyme-substrate complex

Uncompetitive: bind at a site other than active site. Bind only after E-S complex forms, which means increasing [substrate] WILL NOT overcome inhibitor effects. Km decreases, Vmax decreases proportionally. Can exert a positive feedback effect