9-13 Enzyme kinetics

K

Represents the equilibrium constant in enzyme kinetics, reflecting the ratio of products to reactants at equilibrium.

k

The rate constant in the equation that describes the speed of an enzymatic reaction, related to the concentrations of reactants and products.

Km

The Michaelis constant in enzyme kinetics, representing the substrate concentration at which the reaction rate is half of its maximum value.

Enzymes

proteins that lower the Ea of a rxn by providing an alternative reaction pathway, increasing the reaction rate without being consumed.

An enzyme speeds up

both the forward and reverse reactions, decreasing time to reach equilibrium

ΔG+

activation energy

ΔG

difference in energy from reactants to products

ΔG^o’

standard free energy

change at pH 7

Exergonic rxn

spontaneous reactions that give off energy

Endergonic rxn

non-spontaneous reactions that require energy input to proceed.

At equilibrium ΔG

is equal to zero, meaning no net change in the concentrations of reactants and products.

The ΔG of a reaction provides no information about the

rate of the reaction.

in a living cell, reactions which appear unfavorable

can be made to proceed by

removing products rapidly, or by coupling an unfavorable reaction to a

favorable one

Catalase

is an enzyme that catalyzes the decomposition of hydrogen peroxide into water and oxygen, by lowering Ea 3-fold and increasing rate by 10⁸

Carbonic Anhydrase role

catalyze and increase the rate of

conversion of carbon dioxide to carbonic acid

and back again

Carbonic Anhydrase makes a fast reaction

even faster by 10⁶

Cofactors

small

molecules required by

some enzymes for activity, can be coenzymes or inorganic ions and metal

Coenzymes

cofactors that are often organic molecules derived from vitamins

holoenzyme

an enzyme that contains its cofactor and is fully active.

Apoenzyme

the protein part of an enzyme, inactive without its cofactor.

Discovery of enzymes that are not proteins

led to the identification of ribozymes, which are RNA molecules that can catalyze biochemical reactions. Tom Cech

How do enzymes differ from chemical catalysts

Enzymes work under mild conditions
often regulated
stereospecific

Active sites of enzymes create

unique microenvironments

Lock and key model

substrate binds to

that portion of the

enzyme with a

complementary shape

Induced fit model

binding of the

substrate induces a

change in the

conformation of the

enzyme that results in

a complementary fit

Example of induced fit

glucose must be bound to hexokinase before the enzyme alters its shape to initiate the phosphorylation process.
Prevents non-productive ATP hydrolysis

How do enzymes decrease the activation

energy and accelerate reactions?

Transition State (TS) Stabilization

Enzymes can also catayze by bringing reactive groups into

proximity and orientation, facilitating interactions that lower the activation energy needed for reactions.

Acid-Base Catalysis

creating a strong nucleophile reactant by donating or accepting protons, thus stabilizing the transition state.

Nucleophilic Substitutions

Electron rich nucleophile attacks e- poor electrophile

Metal Ion Catalysis

involves metal ions facilitating reactions
Ex carbonic anhydrase

Mechanism of carbonic anhydrase

The zinc ion in the active site promotes the ionization of a bound water molecule

Losing electrons

the process of oxidation, which increases the positive charge of an atom or molecule.

Gaining electrons

is known as reduction, which increases negative charge

Covalent Catalysis

Involves a transient covalent bond between the enzyme and a substrate

examples of covalent catalysis

protein kinase and chymotrypsin

Oxidoreductases

catalyze oxidation-reduction reactions.

(e.g., alcohol dehydrogenase)

Hydro-lyases

cleave bonds with the addition of water.

(e.g., proteases)

Ligases

join two molecules using energy, such as ATP.

(e.g., DNA ligase)

Proteases

catalyze the hydrolysis of peptide bonds, but at different sites

Chymotrypsin is considered

a model enzyme for the study of catalytic mechanisms and is involved in protein digestion.

Chymotrypsin Mechanism involves

acid base catalysis

acyl-enzyme covalent intermediate
hydrolysis

catalytic triad in serine proteases

Serine, Histidine and Aspartic acid work together to facilitate peptide bond cleavage.

Functional groups within the active site

contribute to catalysis

Vmax

is the maximum velocity of an enzyme-catalyzed reaction when the enzyme is saturated with substrate.

chymotrypsin is an example of

non-allosteric behavior

Non-allosteric graph

Hyperbolic

Allosteric graph

Sigmoidal

The Michaelis constant = KM

the substrate concentration at which the reaction rate is half of its maximum value.

KM can be used to help evaluate

specificity of an enzyme

for a substrate (if it obeys M-M conditions)

small Km

indicates high affinity of the enzyme for the substrate.

Big Km

indicates low affinity of the enzyme for the substrate.

Variations in KM have physiological consequences

like aldehyde dehydrogenase, where the weakened mitochondria isoform missing causes symptoms like facial flushing and rapid heart beat while the stronger form is more effective in metabolizing acetaldehyde.

What does kcat mean?

turnover number, because it describes the

number of rxns a molecule of enzyme can catalyze per

second under optimal condition.

What does kcat /KM mean ?

Catalytic Efficiency or Specificity Constant which describes an enzymes

preference for different

substrates

Catalytic perfection

is when kcat/ KM = diffusion rate

A bigger kcat/KM means

the enzyme is more efficient at catalyzing reactions.

Allosteric Enzymes are

“information sensors” that often regulate flux of metabolic pathways

Aspartate Transcarbamoylase

an allosteric enzyme that senses concentrations of CTP to “inhibit” or ATP to “activate” the enzyme

Competitive inhibition in a graph

KM bigger #, =“weaker” substrate affinity 2

Non-competitive inhibition in a graph

decreasing the maximum reaction rate (Vmax) without affecting the affinity (KM) for the substrate. 3

Un-competitive inhibition

occurs when an inhibitor binds only to the enzyme-substrate complex, preventing conversion to product, which decreases both Vmax and KM. 4

Mixed inhibition graph

is characterized by a decrease in Vmax and a change in KM depending on the affinity of the inhibitor for the enzyme and the enzyme-substrate complex. 5

Double-reciprocal (Lineweaver-Burk) plot

where y intercept is 1/vmax

Competitive inhibition double reciprocal plot

non-competitive double reciprocal plot

uncompetitive double reciprocal plot

Mixed competition double reciprocal plot