Biochemistry 1

Front 1

Chemical Kinetics Definition

Back 1

Study of RXN Rates

reactions proceed through an intermediate that is unstable and requires energy to get to that intermediate

Front 2

Energy of Activation Definition

Back 2

Ea= energy required to reach transition state

Front 3

Transition State definition and Example:

A+B ⇋ ABCD ⇋ C+D

Back 3

TS= transition point between reactants and products, it can move forward to produce a product or break up back into reactants

"ABCD" IS the TS

Front 4

How would the rate of a spontaneous reaction be affected if the EA were lowered

Back 4

rate would increase because lowering the EA reduces ammount of energy to reach the TS

Front 5

Catalyst Types and Function

Back 5

Increases the rate of a RXN w/o changing ΔG By:

1) stabilizing the transition state.

2) decreasing the Ea

Enzymes are catalysts that can speed up the binding of substrate and neutralize the charge of the TS

1) must increase RXN rate

2) are regenerated

3) specific to certain reactions

Front 6

ΔG Definition, -ΔG/+ΔG/=ΔG

Back 6

difference in free energy between the reactants and products

-ΔG = spont.

+ΔG = non spont.

=ΔG = Equillibrium

Front 7

Reaction Coordinate With and W/o Catalyst Image

Back 7

Front 8

Describe the Function of Hydrolases & Isomerases and give examples

Back 8

Hydrolases: hydrolyzes bonds (proteases)

Isomerases: rearranges bonds within a molecule to form an isomer

Front 9

Define Reaction Coupling

Exergonic-Endergonic Example:

Exergonic: A+B ⇋ C+D = ΔG-7

+

Endergoinc: W+X ⇋ Y+Z = ΔG +3

=

Back 9

Thermodynamically unfavorable reactions couple with 1 very favorable reaction with free energy

A+B+W+X ⇋ C+D+Y+Z = ΔG-4

Front 10

EX of a favorable reaction

Back 10

ATP hydrolysis, creates ADP and net -12 k/cal ΔG.

Involves Cleaving an inorganic phosphate (Pi) and ADP, produces large ammounts of free energy in the cell because lack of ADP and Pi

Front 11

If a transistion state intermediate possesses a transient negative charge, what amino acids would be found at the active site to stabilize it ?

Back 11

positively charged amino acids such as (His,Arg,Lys)

or

a hydrogen from the -NH2 group of glutamate or asparagine

Front 12

Is it possible for amino acids located far apart in a primary structure of protein sequence to play a role in the formation of the same active site ?

Back 12

Yes, they may be distant 1' but may end up being close in 4' structure

* Illustrates the importance of protein folding and enzyme function

Front 13

If, During an enzyme catalyzed reaction, an intermediate forms in which the substrate is covalently linked to the enzyme via a serine residue, can this occur at any serine residue or does it have to be this specific residue

Back 13

It must occur at the particular serine residue that sticks out into the active site

Front 14

1A) A compound A converts into compound B in solution: A=B, The reaction has the equation constant Keq=[B]Ea/[A]Ea=1000. If pure A is dissolved in H2O at 298 K will Delta G be positive or negative

1B) If pure B is put into a soultion in the presence of an enzyme that catalyzes the RXN b/w A and B what will happen?

Back 14

1A) Solving the equation we see that compound B is 1000 x that of A. If we create a solution with only A it will move spontaneously toward B and have a -Delta G

1B) If only B exists than the back-reaction producing A will predominate until equillibrium is reached.

Front 15

Active Site

Back 15

region cut out of an enzyme that is directly involved in catalysis, requires folding of different substrates to fit

Front 16

Substrates

Back 16

reactants in an enzyme catalyzed reaction, they attach to the enzyme to catalyze reactions

Front 17

Active Site Model Vs. Induced Fit Model

Back 17

Active Site Model: "lock and key" substrate and the active site are a perfect fit

Induced Fit: substrate and active site differ in structure and binding of the substrate induces a conformational change in the enzyme (it must fold to allow substrate to fit)

Front 18

Describe how an Active Site specifies between amino acid and sugar substrates and the configuration in animals

Back 18

-Active sites on enzymes are highly specific in substrate recognition,

1) Stereoisomerism: enzymes catalyzing reactions with monosaccharides are specific for different stereoisomers

2) Amino D and L: enzymes that catalyze reactions involving amino acids are specific for D and L configuration

Animals: L amino acids and D sugars

Front 19

Proteases and how they work on enzymes

Back 19

Proteases are protein cleaving enzymes that have an active site with a serine residue whose OH group acts as a nuceophile, attacking the carbonyl carbon of an amino acid residue on a polypeptide side chain.

EX of Proteases: trypsin, chymotrypsin, and elastase

Front 20

Describe how a Protease Recognition Pocket works and give examples

Back 20

Near the active site on the protease enzyme, the recognition pocket residue attracts certain residues on a substrate polypetide, In which the protease always cuts polypetide chains at the same site (just to one side of the recognition residue)

EX Proteases: Chymotrypsin always cuts on the carboxyl side of the large hydrophobic residues of trp, phe and met

Enzymes that act on hydrophobic substrates have hydrophobic amino acids in their active site

Front 21

Enzyme Function: PH and Temperature

Back 21

Temp: + temp causes peptide motion and hot solution surrounding active site. May disable the substrate, if temps get high enough the protein denatures losing its structure

PH: Several amino acids possess ionizable -R groups that change depending on PH. If the PH deviates significantly it can denature the protein and/or decrease binding affinity of the substrate

Front 22

Enzyme Function: Cofactors and Coenzymes

Back 22

Cofactors: are small metal ion molecules that enzymes require for catalytic activity. Cofactors are inorganic precursors to coenzymes. Vitamins such as B3 are precursors to cofactors such as NAD+.

Coenzymes: are organic molecules of cofactors that bind to substrates during catalyzed reactions. One prime example is Coenzyme A

Front 23

Ways to Regulate Enzyme Activity

Back 23

1) Covalent Modification

2) Proteolytic Cleavage

3) Association with other polypeptides

4) Allosteric Regulation

Front 24

Regulation of Enzymatic Activity: Covalent Modification

Back 24

Phosphorylation of Active Site Amino Acids: Occurs when different groups are covalently attached to active site enzymes to regulate activity

EX: kinase cleavage of a phosphoryl group from an ATP molecule and its addition to hydroxyl groups of serine, threonine and tyrosine. Phosphorylation of these amino acids give them the ability to activate or inactivate the enzyme they are attached too

EX: Protein Phosphorylases: also phosphorylate proteins, but use free floating inorganic phosphate (Pi) in the cell instead of ATP.

Protein Phosphorylation can be reversed by protein phosphotases

Front 25

Regulation of Enzymatic Activity: Proteolytic Cleavage

Back 25

Protease activates, synthesized-inactive enzymes and proteins (zymogens) by cleavage

Front 26

Regulation of Enzymatic Activity: Association with other polypetides

Back 26

Some enzymes have catalytic activity in 1 unit that is regulated by association with a seperate regulatory subunit. Can turn the enzyme on or off

Constitutive Activity: when an enzyme loses its regulatory subunit it demonstrates rapid catalysis

Some proteins require another association peptide just to function

Front 27

Regulation of Enzymatic Activity: Allosteric Regulation

Back 27

modification of an active site through interactions when a molecule binds to an allosteric site on the same enzyme, this alters the conformation of the enzyme allowing [s] to bind

allows the enzyme to be turned on or off

Front 28

Feedback Inhibition of Enzymes: Negative & Positive Feedback (Feedback inhibition)

Back 28

Feedback Inhibition Enzymes act as parts of pathways. Rather than regulating every enzyme in the path, 1 or 2 key enzymes are regulated

Negative Feedback: when excess D is around, shutting off E1 prevents excess B,C, D

Positve Feedback: when [D] is low it activates E1 to stimulate production

Front 29

Describe Reaction Rate of Enzyme Kinetics

Back 29

Reaction Rate (V): ammount of product formed per unit time (mol/s). Dependent on the [S]. If the [S] Doubles so does (V)

Saturation: when the [S] is so high that all of the active sites are constantnly filled

Vmax = point at which enzyme is saturated with[s]/ does not effect reaction rate

Front 30

If there is little substrate in a reaction then?

Back 30

since the rate of V is directly proportional to [S] then the reaction would be slow

Front 31

Michaelis Conastant (Km) describe affinity

Back 31

Km: substrate concentration when the reaction is at half of its Vmax.

Affinity: Km

defines the enzymes level of affinity for a substrate.

Low Km: means the enzyme doesnt need much substrate to get the reaction to 1/2 Vmax. Meaning the enzyme has high affinity for the substrate

Front 32

Cooperativity and Sigmoidal Curve Image

Back 32

1) "Tense State" Low [s] because the enzyme has low affinity

2) "Relaxed State" enzyme substrate affinity increases as more active sites are filled

Front 33

Enzyme Kinetics and Cooperativity

Back 33

1) Binding of the 1st substrate on a multisubunit enzyme allosterically increases the affinity that the other subunit on the enzyme will be filled

2) "Tense State" The conformation of the enzyme before has low affinity for substrate

3) Cooperative Enzymes must have more than one active site, usually multisubunit complexes composed of more than 1 protein structure held together in quateranary strucutre

Front 34

Cooperativity in non enzymatic species

Back 34

Hemoglobin: 4 polypetide subunit protein, with each sub-unit containing a prosthetic heme group, with one O2 binding site

1) O2 binding to one subunit increases the affinity for other units to bind O2

2) When CO2 stabilizes a tense HB it causes all 4 binding sites to have low affinity for O2, giving up any O2 molecules that are still bound

Front 35

Types Enzyme Inhibitors and Def

Back 35

Enzyme inhibitor aim to reduce enzymatic activity.

Inhibitor Types

1) Competititve

2) Non Competitive

3) Uncompetitive

4) Mixed

Front 36

Competitive Inhibition

Back 36

-Molecules that compete w/ substrate for binding on a free enzyme active site

-Inhibition can be overcome by adding more [S] if levels are high enough it will outcompete the inhibitor.

-Competitive Inhibitors require more substrate to reach Vmax

+ in KM because the competition makes it harder for substrate to bind

Front 37

Non-Competitive Inhibition

Back 37

Inhibitors bind only at allosteric sites, and cannot be removed.

SInce the inhibitor fills up one of the sites, less substrate is needed to saturate, thus Vmax is lowered

Km stays the same

Front 38

Uncompetitive Inhibition

Back 38

An inhibitor that can bind to ezyme-substrate-complex only after substrate has bound.

Decreases Vmax, as more enzymes get bound by substrate more inhibitors are able to bind

Km levels decrease because of low inhibition at low [s] at beginning of reaction

Front 39

Mixed Inhibition

Back 39

Occurs when an inhibitor can bind to either unoccupied enzyme sites or enzyme substrate complexes

Free Enzyme: if the enzyme favors the inhibitor with a free active site Km increases

Enzyme Substrate Complex: If an ESC has a higher affinifty for the inhibitor KM decreases

Front 40

The transition state for a reaction possesses a transient - charge. The active site for an enzyme catalyzing this reaction has a HIS residue to stabilize the intermediate. If its residue is replaced with a GLU residue charged at a ph of 7, what effect will this have on the reaction, assuming the reactants are present in excess compared to the enzyme

Back 40

Since His is + charged to stabilize the RXN, a negatively charged GLU would make the enzyme - charged possibly destroying the active site of the enzyme and destabilizing the transition state

Front 41

What is the difference between enzyme concentration and substrate concentration on the Reaction Rate of a catalyzed reaction?

Back 41

Usually the [E] is kept fixed in a reaction. [S] concentration is the only variable that can alter the rate

Substrate concentrations can change much more than Enzyme concentration

Front 42

If a small amount of enzyme in solution is acting at Vmax, and the [S] is doubled, what is the new reaction rate

Back 42

Since it is already saturated their is no rate change

Front 43

Structurally competitive inhibitors resemble what?

Back 43

they resemble the substrate, but the most effective inhibitors resemble the structure of the active site in the TS when it is stabilized

Front 44

If an enzyme has a reaction rate of 1umole/min with a [S] of 50um and another has a rate of

10umol/min with a [S] concentration of 100um does this indicate the presence of a competitve inhibitor

Back 44

no the increase in rate and [S] is at the same ratio so no inhibition is noted

Front 45

Co2 is an allosteric inhibitor of hemoglobin (it dissociates easily when hb is passed through the lungs, Where it is exhaled. CO binds at the O2 site with 300x more affinity than O2, it can be displaced by O2 if O2 concentrations are higher than extracellular CO. Which Statements are correct

1) Carbon Monoxide is an irreversible inhibitor

2) Co2 is a reversible inhibitor

3) Co2 is a non competitive inhibitor

Back 45

1) CO is reversible if O2 ammounts are higher (F)

2) Co2 easily dissociates in the lungs so it is reversible (T)

3) Co2 is non-competitive because it binds at an allosteric site (one that is far from other sites) instead of an active site which would be a competitive inhibitor

Front 46

Which Line is Which Inhibitor

Back 46

Curve 1= unihibited

Curve 2= Non-Competitive

Curve 3= Competitive

Front 47

Allosteric Regulation of Enzymes Image

Back 47

Front 48

Reason for the Enzyme folding into 4'

Back 48

proper formation of the active site

Front 49

Describe Phosporylases and Kinases and provide examples

Back 49

Phosphorylase: transfers a P group to a molecule from inorganic phosphate (Glycogen Phosphate)

Kinase: Transfers a phosphate group to a molecule, from a high energy

Front 50

Gibbs Free Energy Formula

Back 50

ΔG=ΔH-TΔS

ΔG = potential energy

TΔS = Kinetic Energy

Front 51

What is the most important PE storage molecule in all cells

Back 51

ATP, which stores the energy in the ester bonds b/w phosphate groups

Front 52

How Does ATP Hydrolysis Drive Reactions

Back 52

1) conformational change in a protein

2) transfer of a phosphate group from atp to a substrate

Front 53

-ΔH vs. +ΔH

Back 53

-ΔH = exothermic reactions that liberate heat i.e. most metabolic reactions (keeps temp down)

+ΔH = endothermic reactions that require input of heat

Front 54

If the products in a reaction have more entropy (ΔS) than the reactants is equal, can the reactions occur spontaneously?

Back 54

Yes is if ΔS is greater than and ΔH =0

Front 55

Describe Polymerase and Phosphotase and give examples

Back 55

Polymerase: EX DNA polymerase: addition of nucleotides to the leading strand of dna

Phosphotase: removes a phosphate group from a molecule

Front 56

Describe Ligases and Lyases and give examples

Back 56

Ligase = forms a chemical bond (DNA Ligase)

Lyase= Breaks chemical bonds by means other than redox or oxidation

(EX: pyruvate dehydrogenase)

Front 57

Kinase

Back 57

Transfers a phosphate group to a molecule from a high energy carrier such as ATP (EX: PFK)