Biochem Exam 3

Enyzmes

specialized proteins with tertiary or quaternary structure that acts as biological catalysts to speed up biochemical reactions

What are most enzymes?

proteins with a specific three-dimensional or quaternary structure

T or F: each enzyme typically catalyzes a particular reaction or group of related reactions

T

oxidoreductases

transfer of electrons (hydride ions or H atoms)

Transferases

group transfer reactions

Hydrolases

hydrolysis reactions (transfer of functional groups to water)

Lyases

additions of groups to double bonds, or formation of double bonds by removal of groups

Isomerases

transfer of groups within molecules to yield isomeric forms

Ligases

joins molecules together by condensation reactions coupled to ATP cleavage

ΔG^‡

represents free-energy difference between reactants and transition state

T or F: Enzymes are consumed during reactions

F

T or F: Enzymes can be reused

T

What do enzymes change

reaction rate

What do enzymes not change

ΔGº or the reaction equilibrium

What do some enzymes require

cofactors or enzymes to function and may be regulated by other molecules

active site

region where the substrate binds and the chemical reaction is catalyzed

Allosteric site

separate regulatory site where an effector binds, causing a conformational change that increases or decreases enzyme activity

Substrate

reactant acted upon by the enzyme

Product

Molecule formed by the enzyme-catalyzed reaction

effector

a small molecule or protein that binds to a specific protein (like an enzyme or receptor) to increase or decrease its activity

Steps to enzyme catalysis

substrate binds to enzyme

enzyme changes shape to accommodate substrate

a transition state forms as the substrate is being transformed into product

product is formed and is released from the enzyme active site

induced fit

enzyme changes shape to accommodate substrate

Enzymes lower what

activation energy

What must happen to the transition state (EX^‡)

it must be stabilized (lowered free energy) to accelerate the catalysis reaction

Binding EX^‡  more tightly will do what to the energy gap

narrow the energy gap

binding ES more tightly will do what to the energy gap

widen it

What must the enzyme stabilize

EX^‡

the intrinsic binding energy ΔG_b of the enzyme-substrate is partially compensated by

entropy loss that raises the energy of ES

Destabilization (ΔG_d) through strain/ distortion and desolvation

Desovlation of the substrate raises

the energy of the ES complex

De-solvation

charged groups on the substrate lose their stabilizing solvation shell in the active site, making them less stable and more reactive .

Catalytic power

the ratio of an enzymes catalyzed rate of reaction compared to the uncatalyzed rate

Urease catalytic power

10^14

Regulation

mechanisms which control the balance of reactions catalyzed by enzymes, such as in metabolic processes

What does specificity usually result in

near complete conversion of a single product

What illustrates regulation

reduction in rate when a downstream product accumulated illustrates regulation

Specificity

the ability of an enzyme to select for specific substrates (some enzymes have a quite large substrate tolerance as well)

Types of enzyme specificity

bond, group, geometry, stereo, co-factor

Induced fit

(more accurate): active site is flexible, it will change shape to accommodate the substrate

Lock and key

active site is rigid, and once the substrate binds, the enzyme can facilitate the reaction and product release

Sensitivity

how much an environment can or cannot affect the efficiency of an enzyme (including inhibitors)

Cofactors and coenzymes

helper molecules that assist enzymes in biochemical transformations

cofactors

non-protein chemical components required for enzyme activation

coenzymes

organic, non-protein molecules that assist enzymes by carrying chemical groups or electrons (often derived from vitamins)

When are coenzymes typically altered

during the reaction then regenerated

What do coenzymes help to transfer

aldehydes, hydrogen atoms, hydride ion (H-), acyl groups, amino groups, alkyl groups, CO₂, other one-carbon groups such as formyl and methyl groups

Are all coenzymes cofactors?

yes

Are all cofactors coenzymes

no

apoenzyme

the protein portion of an enzyme, typically inactive

holoenzyme

the complete, catalytically active enzyme

Ex.) of inorganic cofactors

Reaction scheme of an enzyme catalyzed reaction

E + S ⇌ ES ⇌ E + P

k1

the binding of the enzyme to the substrate forming the enzyme substrate complex

k2

catalytic rate; the catalysis reaction producing the final reaction product and regenerating the free enzyme (rate limiting step)

which step is the rate limiting step

k2

k-1 or k3

the dissociation of the enzyme-substrate complex to free enzyme and substrate

k-2 or k4

the reverse reaction of catalysis

initial velocity (v0)

the initial rate of the reaction, measured as concentration over time (M/s)

What is initial velocity measured as

concentration/time

Maximum velocity (Vmax)

the maximum rate of reaction when the enzyme is saturate with substrate (concentration much greater that Km)

Is Vmax ever achieved in reality

no

Michaelis constant (Km)

the [S] at which the reaction velocity is half of Vmax

What does Km imply

half of the active sites on the enzymes are filled with substrate and reflects the affinity of the enzyme for its substrate

What does a small Km mean

high affinity even at low [S]

Km (does or does not) depend on enzyme concentration

does not

As [S] increases how much above Km, the reaction velocity approaches Vmax as the enzyme becomes saturated with substrate

well above

What is the steady state assumption

concentration of the ES complex remains constant over time (ES formation = ES breakdown)

zero order substrate concentration

independent of substrate concentration

first order

dependent on one substrate concentration

What shape is the graph of michaelis menten

curved

Michaelis menten equation

Lineweaver-Burk equation

1st

zero

Vmax from lineweaver burk

1/y-intercept

x intercept lineweaver burk

-1/km

y intercept lineweaver burk

1/vmax

Turnover frequency (Kcat)

the number of conversions of S to P over time - allows for the comparison of rate limiting steps and provides information on maximum catalytic rate

Kcat equation

= Vmax / [E]

catalytic efficiency

kcat/Km

measures how effectively an enzyme converts substrate to product- combines speed with binding affinity

Higher catalytic efficiency ratio =

more efficient

Enzyme activity

moles of substrate converted over time - measure of the quantity of active enzyme present under “defined conditions”

specific activity

activity of an enzyme per milligram of total protein - measurement of the purity of then enzyme

Enzyme inhibitor

a substance that interacts with an enzyme and reduces its catalytic activity

Enzyme types

organic or inorganic compounds

Reversible inhibitor is generally classified into how many types

three main

Irreversible inhibition

when an inhibitor binds to an enzyme, permanently disrupting its activity, often forming a covalent bond

What will potent inhibitors do

mimic the transition state

ex of an irreversible inhibition

aspirin acetylating a serine residue in COX-1

Competitive inhibitor

binds at the active site, or partially obstruct the active site

non-competitive inhibitor

inhibitor binds to the allosteric site on the enzyme, away from the active site, changes the confirmation of enzyme and reduces catalytic efficiency

Uncompetitive inhibitor

binds to the enzyme-substrate complex but not the free enzyme

Do uncompetitive inhibitors bind to the free enzyme

no

On lineweaver burk plots where for competitive inhibitors when do lines intersect

on the y-axis

What happens to Vmax in competitive inhibition

it is unchanged

What happens to Km in competitive inhibition

it increases

When do lines intersect in non-competitive inhibition?

x-axis

What happens to Vmax in non competitive inhibition

it decreases

What happens to Km in non competitive inhibition

it is unchanged

In uncompetitive inhibition where do lines intersect

nowhere - they are parallel

What happens to vmax in uncompetitive inhibition

it decreases