Chapter 7: Enzymes

catalyst

change the rate of a rxn by lowering Ea

• doesn’t change during a rxn

• incr rxn rates without being used up, so does not alter equilibrium

enzyme

a substance produced by a living organism that is a catalyst for a biochemical rxn

• a vast majority of biological catalysts are globular proteins (but a few are RNAs)

• does induced fit during the rxn with the substrate

substrate

the ligand for an enzymatic rxn that gets changed by the rxn

product

the result of an enzymatic rxn

active site

the location on an enzyme where a rxn occurs

• very specific for substrates

3 main ways enzymes are used

• diagnosis and prognosis of diseases (by measuring the amount of enzyme in the body)

• as analytical reagents in the measurement of nonenzyme substances (can convert non-detectable molecules to detectable ones like drugs, hormones, etc)

• as therapeutic agents (ex to reopen blood vessels)

mild inflammatory conditions release ____ enzymes

cytoplasmic enzymes

necrotic conditions release ____ enzymes

mitochondrial enzymes

enzymes can incr rxn rates by up to _____ times as fast

x10^19

than the uncatalyzed rxn

rate enhancement

shows how much an enzyme increases the rxn rate

carbonic anhydrase

enzyme that does the carbon dioxide rxn in the lungs

steps of an ezymatic rxn

• enzyme interacts with the substrate, does induced fit

• now you have an enzyme-substrate complex

• a product starts being formed

• the product (changed substrate) gets released from the enzyme

enzymes act in the ____ and ____ rxns

forward and backward

• they speed up both directions of the rxn, helping the rxn reach equilibrium

what types of interactions drive active site binding affinity?

• shape

• charge

• hydrophobics

• any molecular complementarity that is NOT permanent (like covalent bonding)

factors regulating enzyme activity

• pH

• temp

• enzyme conc

• substrate conc

• cofactor and coenzyme conc

1/3 of enzymes are 

metalloenzymes

• require metal ions as cofactors/coenzymes

holoenzyme

a apoenzyme (just an enzyme) with its cofactor/coenzyme/metal ion

• a functional complex

• the enzyme is not functional without its cofactor

coenzymes are often

vitamins that are essential for our diet

some common cofactors

often metal cations

activation energy (Ea)

the energy required to start a rxn

• the difference between energy levels of the ground state and transition state of a rxn

• ΔG‡

• lower activation energy means faster rxn

• higher ΔG‡ means slower rxn

higher ΔG‡ means

slower rxn

ΔG‡S—>P

the Ea for the forward rxn

ΔG‡P—>S

the Ea for the reverse rxn

ΔG‡°

the energy difference between reactant and products

• determines if a rxn is spontaneous

free energy ΔG

the amount of energy available to do work

in spontaneous reactions, _____ have less free energy than ____

products have less free energy than the reactants (so the forward rxn is favored)

ΔG will be neg (exergonic rxn)

even if ΔG° is neg, a rxn can be slow if

there is a lot of Ea

‡

transition state

positive ΔG means

the rxn is not spontaneous

• backwards rxn is favored

• endergonic rxn

if ΔG=0,

the reverse and forward rxn are equally favored

ΔG is the energy difference between

the substrate and the product

enzymes lower the

Ea and therefore the energy difference between the substrate and the transition state

NOT ΔG (which is the energy diff between the substrate and the product)

more Ea means

slower rxn

catalysts reduce

the Ea of a rxn

ΔG‡

free energy of activation

• the energy required to convert 1 mol of substrate from the ground state to the transition state (‡)

the more molecules reaching the transition state means

the more likely product forms and therefore the faster the rxn reaches equilib

what binds better to the enzyme?

the transition state (of the substrate)

• otherwise the rxn would not occur

• it enables the product to actually be made since the substrate changes into the transition state so that it can bind to the enzyme better

ways that enzymes are catalytic

• proximity effect: bring substrates and active sites together

• orientation effect: hold substrates at the exact distance and orientation necessary for rxn

• catalytic effect: provide basic/acidic/etc side chain groups required for catalysis

• energy effect: lower the energy barrier by inducing strain in bonds in the substrate molecule

how do enzyme-catalyzed rxns begin?

with the migration of the substrate into the active site to form an enzyme-substrate complex

• mediated by weak noncovalent interactions and shape

• induced fit can cause both the enzyme and the substrate to change shape

  • water can be displaced by the binding of the enzyme to the substrate

catalytic effect mechanisms of enzymes

• acid/base catalysis: by removing/adding a proton to/from something to lower the free energy of the transition state

• covalent catalysis: when the enzyme covalently binds to the substrate for a brief time (transiently) to initiate the formation of the enzyme-substrate interaction

• metal ion catalysis: metal ions participate in the rxn mechanism

• many enzymes use a combination of these methods

acid base catalysis

• a proton transferring from an acid lowers the free energy of the rxn’s transition state (general acid catalysis)

• a proton being removed by a base lowers the free energy of the rxn’s transition state (general base catalysis)

• these rxns occur to stabilize intermediates to promote the continuation of a rxn

• see image on slide

• the substrate has a peptide bond

• the intermediate has a charge (since when the enzyme and substrate combined, it was due to a proton transfer)

• the proton transfers to and from either water or weak acids/base side chains to stabilize the intermediate, but this causes the peptide bond to be unstable

• the product forms when the peptide bond is finally broken due to a leaving group leaving

amino acids that serve as proton donors/acceptors based on protonation

• Glutamic acid

• aspartic acid

• lysine

• cysteine

• histidine

• serine

• tyrosine

• proton donors are in protonated form (have extra H, but aren’t always +)

how enzymes serve as covalent catalysts

• a transient (temporary) covalent bond forms between the enzyme and the substrate

• requires a nucleophile on the enzyme

nucleophiles that can be on an enzyme for a covalent catalysis

• reactive serine

• thiol

• amine

• carboxyl

metal ion catalysis

• when there is a coenzyme-like metal ion bound to the enzyme, which interacts with the substrate to facilitate binding of the substrate to the enzyme

• metal ions help:

  • orient substrate for rxn

  • stabilize neg charges (since they’re often pos)

  • mediate redox reactions (keep other ions in certain charges)

chymotrypsin is a 

protease

chymotrypsin rxn pathway in the GI tract (ex of acid base covalent catalysis)

• the protease chymotrypsin cuts off aromatic side chains in a peptide

• His acts as base and Ser acts as the acid

• peptide bond between C=O and HN (blue line)

• N from His grabs the H from Ser’s OH (so His acts as base)

• O of Ser attacks C of the C=O by the peptide bond of the substrate’s side chain, covalently attaching to it

• now C-N bond (peptide bond) is destabilized, so N takes the H from His, so now N of the substrate broke the peptide bond

• now part of the substrate is not held into active site, and it moves out

• Ser is still covalently attached to the substrate in the active site of the enzyme

• water comes in and breaks the Ser-substrate covalent bond so the substrate can leave

• active site gets regenerated

exergonic rxns are essential because

you need to counteract entropy

since cells cannot tolerate rxns that release huge amounts of energy/heat is released, what does it do instead?

many smaller rxns in a series of steps

ex: in glycolysis

types of enzymes

oxidoreductases: transfer electrons using H atoms of hydride ions

transferases: transfer the groups between two molecules

hydrolases: do hydrolysis (transfer functional groups to water)

lyases: cleave C-C, C-O, C-N or other bonds by elimination, leaving double bonds or rings, or add groups to double bonds (addition)

isomerases: transfer groups between molecules to yield isomeric forms

ligases: form C-C, C-S, C-O, and C-N bonds by condensation rxns coupled to cleave ATP or a similar cofactor

translocases: move molecules/ions across a membrane

oxidoreductases

catalyze e- transfer in redox rxns

• usually uses energy carriers like NAD+ to do redox rxns

transferases

transfer a functional group from one molecule to another so that they switch groups

hydrolases

cause the cleavage if bonds using water (to break them apart)

• aka do hydrolysis

• includes proteases

lyases/synthases

cleave or form a bond between two molecules (without using water or doing redox)

• synthases do the forward rxn

• liyases so the reverse rxn

isomerases

move groups within a singular molecule

• intramolecular rearrangement to make a new molecule

ligases/synthetases

join two substrates together by cleaving ATP or another energy molecule

• uses nucleotides for energy

translocases

move molecules or ions across membranes

mechanism for enzymes that involve a ternary complex

can be ordered or be random

• ordered: E must bind S1, THEN S2 before forming products (P1 and P2)

• random: E can bing S1 and S2 in any order, but once both are bound to E, the products (P1 and P2) are formed in some manner

mechanism for enzymes that do NOT involve a ternary complex

the order matters

• E binds S1 and forms P1 then binds S2 and makes P2

• E gets changed after it makes P1, causing it to now bind S2

kinetics

the study of the rate at which compounds react

• developed by Michaelis and Manten

velocity in kinetics is

the speed of the rxn

at low [S], v incr almost linearly with

an incr in [S]

• but when [S] is already high, v increases by smaller and smaller amounts in response to an incr in [S]

vmax

the point at which increasing [S] does not increase velocity any further, so the graph plateaus

rxn rate depends on

substrate conc

units ion velocity for enzymatic rxns

molarity/time

[Product]/seconds or minutes

velocity incr as [S] increases in a ____ manner

hyperbolic

can you use the vmax of two enzymes to compare them?

NO

• vmax is only proportional to the conc of enzyme, so cannot be used to compare across different enzymes

turnover number (kcat)

the number of substrate molecules converted to product per enzyme per unit of time

• only when E is SATURATED with substrate

kcat= Vmax/[E]

has units of /s or /min

Vmax=

[E] (Kcat)

two enzymes catalyzing different reactions could have the same

kcat

• but their rates of their uncatalyzed rxns are likely different

• so their rate enhancements likely vary greatly

Michaelis-Manten Constant

Km= [E][S]/[ES]

km is a

constant

• since once the rxn starts, [ES] is constant

• [ES] can break down and do reverse rxn too

steady-state assumption

idea that the rate of formation of ES is equal to its breakdown

• so [ES] is constant

enzymes can have diff ____ for diff ___

Kms for diff substrates

steady state assumption equation

k1[E][S]= k-1[ES]+K2[ES]

basically Km=[E][S]/[ES]

a small Km indicates

tighter binding

• since less substrate is unbound

• Km= [E][S]/[ES]

Km is

the [S] at which half of the enzyme molecules have substrate bound in their active sites to produce ES

• so Km=[S] since [E]=[ES]

Km is inversely proportional to

how well an enzyme binds its substrate

does km equal ½ Vmax?

NO

• Km is the [S] at ½ Vmax

Michaelis-Manten equation expressed in terms of Vmax and Km

Vo= [(Vmax)[S]]/Km + [S]

• this equation graphs as a hyperbola

Vmax is the velocity of the rxn when

substrate is infinitely available

• so [S]>Km

when [S]=Km, vmax is

at ½ vmax

catalytic efficiency

kcat/Km

• since affinity is inversely proportional to Km and kcat reflects rate of formation

a perfect enzyme would have

a high kcat/Km ratio

• since you want Km low and kcat high

• few enzymes are “perfect”

why is enzyme inhibition helpful?

• for regulation

• don’t want enzymes active all the time

many drugs and toxic agents act to

inhibit enzymes

AIDS treatment

• scientists are trying to develop specific inhibitors to block the enzymes that are unique to HIV

• integrase: inserts the viral genome into the host genome

• protease: cuts up proteins into smaller pieces to be replicated

• reverse transcriptase: turns RNA into DNA

• these are the 3 enzymes used by the HIV virus that you want to inhibit

enzyme inhibitors decr

the activity of the enzyme

irreversible inhibitors (inactivators)

• covalently attach to the enzyme so that substrate cannot bind

• often are powerful toxins

• ex: penicillin is an antibiotic that reacts with the active site of a bacterial enzyme to inhibit it so no more bacterial cell wall can be made and the bacterial cells stop replicating

reversible inhibitors

• bind to the enzyme reversibly (so can easily dissociate, therefore don’t covalently bond but use molecular complementarity stuff)

• are often structural analogs of substrates or products that they are replacing/inhibiting

• can bind to the free enzyme to prevent it from binding to the substrate OR bind to the ES complex to prevent it from creating the final product

• used as drugs to slow down an enzyme

2 types of reversible inhibitors

competitive and not competitive

competitive: when the inhibitor beats out the substrate to bind to the enzyme at the active site

Not competitive: when the inhibitor interacts with another spot of the enzyme (not the active site) OR does both

what types of inhibition affect the Km?

Competitive, uncompetitive, and mixed types of enzyme inhibition affect the apparent Km (Michaelis constant), while non-competitive inhibition typically does not

• competitive: incr Km by decr substrate binding affinity

• uncompetitve: decr Km by incr the enzyme’s affinity for the substrate

• mixed: can either increase or decrease apparent Km, depending on whether the inhibitor preferentially binds to the free enzyme or the enzyme-substrate complex

• non-competitive: binds equally to the free enzyme and the enzyme-substrate complex, causing the Km to remain unchanged

competitive inhibition

when the inhibitor binds in place of the substrate in the active site, reducing the productivity of the enzyme

• the inhibitor does not affect the catalysis of substrate forming product

• if [S]>[I], enzyme will bind more preferentially to the substrate than the inhibitor

see how substrate conc affects competitive inhibition

the inhibitor acts as a transition state analog

if [S]=[I], then the inhibitor or substrate bind 50/50

when more substrate gets added, it binds more preferentially than the inhibitor

methotrexate

• a competitive inhibitor and chemo drug

• competes with dihydrofolate (the substrate) to inhibit nucleotide synthesis

• basically outcompetes the dihydrofolate so that new nucleotides cannot be made by the enzyme and slows down cell division for a cancer pt to defeat the tumor

• but also leads to side effects since your other cells can’t divide either, makes you feel ill

dihydrofolate

can be converted into nucleic acids for replication

competitive inhibitors’ affect on Km

incr Km, since the inhibitor decr enzyme affinity for substrate (so more substrate is needed to obtain the same rxn rate)

but all values still reach the same vmax, which is unchanged

Lineweaker-Burk plots

• can be used to distinguish between diff types of inhibition

• the slope of the line shows Km/Vmax (which is the inverse of the Michaelis-Manten equation)

• the y-intercept is 1/Vmax

• x intercept is -1/Km

• plot shows unhibited rxn

Lineweaver-Burk plot for competitive inhibited rxn

• slope (Km/Vmax) incr as [I] incr

• intercept on x-axis (-1/Km) changes to show an increased Km when the inhibitor is present

• the Vmax remains unchanged

• slope incr as [I] incr

uncompetitive inhibition

• the inhibitor binds to the whole ES complex, to stop it from forming product

  • the ESI complex can NOT form products

• the binding site for the inhibitor isn’t created until the enzyme binds to the substrate

• inhibition can NOT be overcome by adding more S