BIOCHEM: Enzyme Kinetics and Regulation

Gibbs Free energy (G)

- the energy available to do work (chemical rxns)

- Positive delta (G) : non-spontaneous rxn

- Negative delta (G) : spontaneous rxn

Enzyme affect on G

- enzymes lower the activation energy, esulting in a net negative delta G rxn

-enzymes dictate how the activation energy will be lowered

exergonic

- (-) delta-G

- rxn is spontaneous

- products end lower than reactants

endergonic

- (+) delta-G

- rxn is non-spontaneous

- products end higher than reactants

kinetics

- the formation of product overtime or the consumption of reactant over time until equilibrium is reached

Formula for rxn rate

V = -delta(S) / delta(t) (reduction in reactant over time)

V = delta(S) / delta(t) (increase in product over time)

rxn rate depends on [S]

- changing [S], changes rxn rate

- slope at initial is linear

Michaelis Menten Plot

Vo vs. [S]

Vmax:

max rxn rate once all enzymes are saturated

1/2 Vmax:

rxn rate at which half of all enzymes are saturated w substrate

Km:

a constant that describes the [S] web rxn is at 1/2 Vmax

V:

rxn rate at a given [S]

Michealis-Menten Equation

V = (Vmax x [S]) / (Km + [S])

When [S] << Km

- rxn rate is directly proportional to [S] (linear)

- as substrate increases, the rxn rate will increase proportionately

When [S] >> Km

- rxn rate is no longer directly proportional to [S] (non-linear)

enzymatic breakdown of ethanol in liver:

- ADH (Alcohol dehydrogenase)

- ALDH (Aldehyde dehydrogenase)

- ethanol (ADH) --> acetaldehyde (ALDH) --> acetate

isoenzymes

- catalyze the same rxn, but have structural/chemical differences leading to different activity (Km)

- Two main ALDH isoenzymes in liver: mitochondrial ALDH (low Km) and cytoplasmic ALDH (high Km)

Mitochondrial (ALDH) vs. cytoplasmic (ALDH) Km's

- low Km: (m) conversion of acetaldehyde [S] to acetate [P] is occurring at a high rate at low [S]

- high Km: (c) conversion of acetaldehyde [S] to acetate [P] is occurring at a low rate at low [S]. it takes higher conc. of acetaldehyde to increase rxn rate

Alcohol Flush Syndrome

- mutation in mitochondrial ALDH leaves only cytoplasmic ALDH fxnal

- High km bc of cytoplasmic ALDH and this converts acetaldehyde into acetate slowly, causing buildup of acetaldehyde and it escapes into blood

enzyme regulation

- enzymes are energetically expensive for the cell to produce so activity can be decreased by negative regulators instead of destroying them

reversible inhibitors

- bind to enzyme to reduce activity, but can rapidly dissociate in the right condition, restoring enzyme activity quickly (competitive and non-competitive)

irreversible inhibitors

- bind strongly to enzyme to reduce activity; dissociates slowly, typically new enzymes must be made to restore significant activity

- not made in the body; some drugs and toxins

irreversible inhibitor ex.'s

- aspirin binds irreversibly to cyclooxygenase

- penicillin binds irreversibly to bacterial trans peptides

- cyanide binds irreversibly to cytochrome C oxidase

competitive inhibition

- inhibitor blocks active site and prevents formation of enzyme-substrate (ES) complex

noncompetitive inhibition

- (allosteric) inhibitor binds to ES complex to reduce the rate of rxn

uncompetitive inhibition

- binds to ES complex after substrate has bound to prevent enzyme from releasing product

Kinetics of competitive inhibition

- on plot: no change in Vmax, increased Km = more substrate needed to reach 1/2 Vmax

- it takes more substrate to reach Vmax when it is present

- reduces rxn rate by reducing how much substrate can bind to enzyme but can be reversed by addition of [S]

Kinetics of noncompetitive inhibition

- on plot: reduction in Vmax, no change in Km

- slower rxn rate (shallow slope) and reduction in max rxn rate

- does not block active site and can not be reversed by increasing [S]

Enzyme positive regulation types:

1. allosteric activation (control)

2. reversible covalent modifications

3. proteolytic activation

1. Allosteric control

- binding small molecules in enzyme regulatory sites

- allosteric inhibitors and actiatiors

allosteric inhibitors

- induce a conformation change in the active site to prevent substrate binding

allosteric activators

- induce a conformation change in the active site that allows substrate binding

2. Reversible covalent modification

- process involves the reversible covalent linkage of fxnal groups to an enzyme that will modify its activity

2. Reversible covalent modification ex.'s

ex. phosphorylation / dephosphorylation (adds or removes phosphate group) and acetylation / deacetylation (adds or removes acetyl group)

phosphorylation and dephosphorylation

- protein kinases catalyze the addition of phosphoryl groups

- phosphoryl can be accepted by OH groups (ex. Ser, Thr, Tyr)

- phosphates catalyze the removal of phosphoryl groups via hydrolysis, producing an OH group and a phosphate group

ATP donates...

- Adenosine triphosphate (ATP) donates phosphate groups

- ATP is produced by metabolic processes, connecting enzyme activity to the metabolic state of the cell

3. Proteolytic activation

- some enzymes are synthesized in an inactive state (zymogen) and must me proteolytically processed in oder to become active

- helps ensure enzymes are only activated when they reach correct region of cell or tissue or when they are needed

Why is proteolysis important?

- important for fxnal digestive enzymes, blood clotting factors, transitions btwn developmental stages, apoptosis

proteolytic activation ex.

Chymotrypsin is cleaved by enzyme Trypsin to become activated:

1. first cleavage by trypsin

2. second cleavage through autocatalysis

3. three chains are connected by disulfide linkages