biochem midterm 2

biol 100 @ ucsc w/ joshua arribere

what is the first law of thermodynamics?

states that energy cannot be created or destroyed, only transferred or converted from one form to another.

total amount of energy in universe remains constant though the form of the energy can change

what is the second law of thermodynamics?

the universe constantly changes to become more disordered

what is enthalpy?

referred to as H, measure of energy in a system

releases energy as heat

stored in biochemical bonds that can be broken to release heat

what is entropy?

referred to as S, measure of order in a system

as energy is transferred or transformed, it tends to increase, leading to a decrease in the amount of available energy to do work.

only reactions that cause a decrease in available energy (delta G is negative) will occur

spontaneously

many biochemical reactions that occur in cells would not occur spontaneously at the temp at which cells live, so they use _________ to drive these reactions

catalysts

most catalysts are:

proteins and referred to as enzymes

when energy in chemical bonds are released, they:

generate heat or do work

in the absence of an input of energy, all reactions:

tend spontaneously towards a greater state of disorder

creating order within cells requires an input of energy, which results in:

a greater increase in disorder elsewhere in the universe

the gibbs free energy equation defines

the maximum amount of work that can be obtained from a system at constant temperature and pressure, also if a reaction occurs spontaneously

what two main factors contribute to the large release of free energy when ATP is hydrolyzed?

electrostatic repulsion and resonance stabilization

what is electrostatic repulsion?

ATP contains two phosphoanhydride bonds. the oxygen molecules surrounding each phosphate have a partial negative charge. hydrolysis of ATP releases this

what is resonance stabilization?

terminal phosphate of ATP has fewer resonance states available than free phosphate. when ATP is hydrolyzed, the released phosphate has more of this

what does delta G depend on?

energy stored in chemical bonds and concentration of reactants

when delta G < 0 , the reaction is:

spontaneous

when delta G > 0 , the reaction is:

non spontaneous

when delta G = 0 , the reaction is:

in equilibrium

when Keq < 1, what is delta G?

delta G > 0

when Keq > 1, what is delta G?

delta G < 0

when Keq = 1 , what is delta G?

delta G = 0

for the reversible enzymatic reaction X<>Y, delta G is 15 KJ/mole. if we set the concentrations of X and Y each at the same concentration, what would happen?

more reactant will be made

for the reversible reaction A<>B, K’eq = 2.0. if we set the concentration of substrate A at 2.0 M and the concentration of product B at 1.0 M, what would happen?

more product is made

true or false: the enzyme gets changed in the reaction it catalyzes

false

does changing the activation energy change delta G?

no, because activation energy relates to the rate of a reaction which does not affect delta G.

how does enzyme catalysis affect the activation energy of a reaction

lowers activation energy

how does enzyme catalysis affect the delta G of a reaction

does not affect delta G

how does enzyme catalysis affect the equilibrium of a reaction

does not affect equilibrium

how does an enzyme lower the activation energy of a reaction

it binds and stabilizes transition state, which lowers activation energy

for a reversible reaction that is catalyzed by an enzyme and is in equilibrium, what is the effect of additional enzyme added to the reaction?

no effect, since enzyme does not affect free energy

for a reversible reaction that is catalyzed by an enzyme and is in equilibrium, what is the effect of additional product

reaction is reversed, increased free energy on product side, then reaction will want to increase the free energy on the other side

what is kcat?

the turnover number or catalytic constant of an enzyme, which represents the number of substrate molecules converted to product per unit time by a single enzyme molecule when it is fully saturated with substrate.

hydrolase

uses water to break a chemical bond

nuclease

cleaves nucleic acids

protease

cleaves proteins

kinase

add one or more phosphates

phosphatase

remove one or more phosphates

GTPase

hydrolyze GTP

ATPase

hydrolyze ATP

true or false: delta G depends on the concentration of reactants and products

true

true or false: delta G is constant

false

true or false: delta G measures the rate of a reaction

false

true or false: an increase in entropy means that delta G will be positive

false

what is the first assumption of the michaelis menten equation?

the formation of the enzyme-substrate complex is the rate-limiting step in the overall reaction, ignore product binding to enzyme

what is the second assumption of the michaelis menten equation?

the rate of formation of the enzyme-substrate complex is much faster than the rate of breakdown of the complex, steady state assumption, \[ES\] is constant

for an enzyme with a very low Kcat, what will Km be approximately equal to?

Kd

what is an assumption of michaelis menten kinetics?

\[ES\] is constant

what is Km?

concentration of substrate required for 1/2 maximal reaction velocity 

when Kcat is very low, Km approaches the:

Kd

Kcat/Km is a measure of:

the overall efficiency of an enzyme and has a theoretical limit of 10^8 to 10^9 sec^-1 M^-1

limit is imposed by diffusion since an enzyme can only make product as fast as it can collide with a molecule of substrate

what is an abzyme?

an antibody that has catalytic activity, meaning it can act as an enzyme.

covalent catalysis

active site contains a reactive group (usually powerful nucleophile) that forms a transient covalent bond during catalysis

example of this is lysozyme

acid/base catalysis

molecule in the active site acts as a proton donor or acceptor

in some cases both an acid and a base can carry out functions in active site

example would be proteases that cleave peptide bonds

metal ion catalysis

(…………) can serve as electrophilic catalyst, can create a nucleophile by increasing acidity of nearby acid, can contribute an ionic interaction that facilitates substrate binding

orientation and increasing concentration

enzymes bind two or more substrate molecules, which can increase the local concentration of the molecules to facilitate reaction, and orient molecules for reaction

example of how substrate binding contributes to catalysis

negative feedback

product of pathway binds and inhibits the enzyme at the top of the pathway

to maximize speed and efficiency of reactions in metabolic pathways, many enzymes work as

large multi enzyme complexes

true for metabolic enzymes, but also for enzymes that catalyze synthesis of RNA, DNA, and protein

reversible inhibition

inhibitor binds to enzyme but inhibition is reversible, as the inhibitor can dissociate from the enzyme leaving it unchanged

inhibitor must be present at relatively high concentrations to inhibit enzyme

irreversible inhibition

inhibitor reacts either covalently with key functional group in active site to inactivate the enzyme, or it binds very tightly to be irreversible

reversible inhibitors have 3 types

competitive, non competitive, and substrate-dependent non competitive inhibition

competitive inhibition

inhibitor resembles the substrate and competes for binding of substrate to the active site

directly bind to active site and prevent substrate from binding

can be overcome by addition of higher concentrations of substrate

what happens to Km in competitive inhibition

increases Km, since more substrate is needed to achieve a higher velocity

what happens to V max in competitive inhibition

does not affect V max

non competitive inhibition

inhibitor binds to site other than active site and causes a conformational change at the active site that decreases activity

cannot be overcome by adding more substrate

what happens to Km in non competitive inhibition

does not affect Km

what happens to V max in non competitive inhibition

V max is decreased

substrate dependent non competitive inhibition

inhibitor only binds to enzyme when it is bound to the substrate

lowers Km and V max

true or false: for an enzyme reaction in which \[S\] is much higher than Km, nearly all \[E\] active sites have substrate bound

true

true or false: for an enzyme reaction in which \[S\] is much higher than Km, the rate of the reaction is limited by K(-1)

false

true or false: for an enzyme reaction in which \[S\] is much higher than Km, the rate of the reaction is limited by K(1)

false

true or false: for an enzyme reaction in which \[S\] is much higher than Km, the reaction rate is less than 1/2 V max

false

true or false: for an enzyme reaction in which \[S\] is much higher than Km, the reaction rate is not determined by K cat

false

what would be the effect of a mutation that disrupts catalytic mechanism (conversion of substrate to product) but not substrate binding

decreased Km

decreased V max

DNA polymerase is an example of what kinds of catalysis

metal ion catalysis

orientation and increasing concentration catalysis

ligand often refers to

a small molecule such as ATP, cAMP, or a hormone, that binds to a protein

can also refer to a macromolecule, including protein, DNA, or RNA

__________ binding interactions between molecules are essential for virtually all biochemical processes

non covalent

in complex cases, binding of one ligand molecule can cause

conformational changes in a protein that affect binding of a second ligand molecule

referred to as allosteric binding

how are dissociation constants measured experimentally

measuring the amount of ligand bound to protein

what is a method used to measure the amount of ligand bound to protein

one can add increasing amounts of radioactively labeled ligand (such as drug/hormone) to solution of protein

after binding, solution is passed through nitrocellulose filter

then free protein and protein bound to the ligand stick to nitrocellulose filter, whereas the ligand passes through

final step is measuring the amount of radioactivity that sticks to the filter

what happens after ligand binds to protein in one of the methods used to measure the amount of ligand bound to protein

the solution is passed through a nitrocellulose filter

then free protein and protein bound to the ligand stick to nitrocellulose filter, whereas the ligand passes through

in the common method used for measuring the amount of ligand bound to protein, what is the final step

measuring the amount of radioactivity that sticks to the filter

in the common method used for measuring the amount of ligand bound to protein, what is necessary

using a small ligand that does not bind to nitrocellulose

what does the value of the dissocation constant determine

ligand concentration range over which the protein switches from bound to unbound

what Kd value does the drug molecule need to have a high affinity for the target to be useful

between 10^-9 to 10^-12 M

what happens if the drug has a low affinity for their target

they must be used at relatively high concentrations to reach the significant occupancy of their target binding site

if a drug with low affinity for their target is being used at a relatively high concentration then what is more likely to happen

the drug will undergo “off target” interactions with other proteins for which it has a weak affinity

what do off target interactions do

can cause dangerous side effects

what minimizes the chance of off target effects

drugs having a high affinity for their target so they can be used at a very low concentration

how are most drugs developed

optimizing inhibitors of a protein target

to inhibit a protein what function does it need to have

needs to have a function that is “druggable”

enzymes are considered druggable as they have a small active site that can be inhibited by the binding of a small molecule

what are compounds isolated from living organisms referred to as

natural products

natural products

have the advantage of having greater complexity

advantage of their structures being biased towards having biological functions

lead compounds

molecules that are first discovered in the search for a drug that is not usually ideal for the properties needed for a useful drug

how are lead compounds optimized

optimized by chemical modification to increase their affinity, solubility, stability, etc

imatinib

revolutionary anti cancer drug that dramatically improved affinity and specificity

how does imatinib work

binds to the ATP binding site of the BCR-ABL kinase and prevents ATP from binding

why are proteins that contain binding pockets for their substrates or ligands good targets for drugs

their binding pockets make excellent high affinity binding sites for drugs

binding of drugs to their target proteins results in:

conformational changes in the protein

in the same way that substrates often induce a change in the conformation of an enzyme, referred to as induced fit

induced fit

drug or substrate binds preferentially to one of many conformational states of a protein, and then stabilizes that conformation

conformational selection

drug or substrate binds most effectively to a limited number of the many conformations that a protein adopts

will often bind to high energy conformation that occurs rarely in the absence of ligand