Biochem - Section 2: Enzyme Kinetics and Inhibition

enzymes

molecules that act as a catalyst in a reaction

not all proteins are enzymes and not all enzymes are proteins

Catalyst

act in a reaction to decrease the amount of activation energy need to reach the transition state of a reaction

changes the rate of the reaction but not the change in free energy

What do enzymes do to ΔG‡

they decrease it

What do enzymes do to ΔG

nothing they don’t change this

what do enzymes do to Keq

ezymes do not have an effect on the equilibrium constant of the reaction

How do enzymes quicken the rate of the reaction

they bind to the transition states of the reaction better than substrates

enzyme bind to ES than then tighter binding to the transition state stabilizes the interaction and lower the activation energy needed

read this reaction coordinate diagram

ΔG‡ = activation energy

ΔG = change in gibbs free energy

what factor do the change of free energy of a reaction rely on

standard change in free energy

concentrations of reactants and products

note tat this is additive

nucleophiles and electrophiles

nuc: electron rich moelcule that donates pair of e- to form a bond

el: electron poor molecules that accept a pair of electron to form a bond

acid base catalytic mechanism

giving and taking protons

covalent catalysis

involves a change of reaction paths

metal ion catalysis

uses redox factors or pKa shifters

electroststic catalysis

preferential interaction with TS

oxidoreductases

class of enzyme used in oxidation-reduction reactions

transferase

class of enzyme used for the transfer reaction of functional groups

hydrolase

class of enzyme used in reactions where H2O is used to break a bond

lyase

class of enzyme used in reactions where functional groups are added or removed

isomerase

class of enzyme used in reactions where compound is converted to its isomer

ligase

class of enzyme used when a new bond is formed that requires energy

homolytic cleavage

rare cleavage of a bond where the electron pair splits - one goes to the 1st product one goes with 2nd product

heterolytic cleavage

more common cleavage where both electron go to the same product

leads to unstable products

pre steady state

phase of enzyme reaction where the [ES] is building up but is not steady -see green area

Steady state

[ES] is constant, and P forms at a steady rate - see orange area

this is the phase modeled by the Michaelis-Menten kinetics models the steady state

Approach to equilibrium

S is depleted and the rate slows

initial velocity (V0)

the slope of the tangent to the progress curve at t=0

this is measured in the steady state of the reaction - when [S] » [E]

each starting [S] give one Vo;

Does V0 increase linearly with [S]

No it does not

there are only so many enzyme active sites

the enzyme eventually saturates which means that all sites are busy so more substrate doesn’t raise V0

Km

Michaelis constant - an approximate measure of substrate’s affinity for an enzyme

units - mM, micromole

lower Km -half of Vmax is reached at lower [S]

higher Km - more [S} is needed to reach half of Vmax

kcat

turnover number - the number of substrate molecules that an enzyme molecule can convert per second

kcat = Vmax/[E]

units - s^-1

Vmax changes with [E], kcat doesnt

assumptions of MM kinetics - 5

1. initial velocity is measured early so [P] is low and E + P → ES is ignored

2. [S] » [E] ([S] = [Stotal]

3. in the steady state, [ES] is constant (the rate of formation equals the rate of breakdown)

4. theres a constant amount of enzyme present

5. the scheme is simple - one substrate, one product, independent active sites

MM scheme

E + S → ES → E + P (there a k1, k-1, and k2 all are rate constants)

rate of product formation depends on [ES]: V0= k2[ES]

higher [S] = more ES and a faster reaction

Vmax

the rate of reaction when [S] increases to the point where all active sites are occupied (saturation)

Vmax = kcat[Etotal]

units - micromoles/min

how are Km and Vmax related

Km is ½ of Vmax

kcat/Km

measures how efficiently an enzyme uses S at low [S] (catalytic efficiency)

units - M^-1s^-1

limit of the result is how fast S diffuses to the enzyme

perfect enzymes - 10⁷ - 10⁹ M^-1s^-1

when does Km = Kd (dissocication)

only when k2 « k-1

what kinds of kinetic mechanisms can multi-substrate reactions have

sequential mechanisms

ping pong mechanisms

sequential mechanism

binding creates a tertiary complex where all substrate must bind to the enzyme before product is released

can be random or ordered

ping pong mechanism

substrate bind and release before the next substrate comes in

no tertiary complex is involved

Lineweaver-Burk plot

plots 1/[S] vs 1/Vo

slope - Km/Vmax

y interept - 1/Vmax

x-intercept = -1/Km

Why are lineweaver plots less accurate

the plot cluster points and distorts the error bars so that small errors at low [S] become large errors in 1/V0

a V0 vs [S] plot has error bars of equal sizes for each [S]

irreversible inhibitors

react with the enzyme

one molecule can permanently shut off one enzyme molecule

powerful toxins but may be used in drugs

reversible inhibitors

bind to and can dissociate from the enzyme

can be structural analogs of substrates or products

often used as drugs to slow a specific enzymes

can bind to free E to prvent binding to S or bind to ES and prevent the reaction

Penicillin

acts as a irreversible inhibitor

ressembles the substrate and binds to the active site of the enzyme

it forms a covalent bond with the enzyme and makes it so the enzyme cannot build the bacteria cell wall

competitive inhibition

type of reversible inhibition where the inhibitor directly competes with the substrate making it so it cant bind

does not affect catalysis directly or react to form P

Vmax stays the same, but the apparent Km increases

competitive inhibition lineweaver plot

slope increases with increasing inhibitor concentration

lines meet on the y axis

y intercept = same , X int = moves toward 0

uncompetitive inhibitors

binds only the ES complex at an allosteric site and prevents catalysis, no effect on substrate binding

Vmax and Km both decrease

uncompetitive inhibitor lineweaver

slope remains the same - cause Km/Vmax stays the same

y intercept increases making Vmax smaller

parrellel lines

mixed inhibition

binds to E or ES at a different site than active site so substrate binding and catalysis are inhibited

affinity for E and S differs

Vmax decreases, apparent Km can increase or decrease

mixed inhibition lineweaver-Burk

slope and the y intercept both increase

x int moves either way

lines meet left of the y axis

Noncompetitive inhibition

special case of mixed inhibition where it binds to E and ES with the same affinity

inhibitor binds to a different site than the active site

it blocks catalysis and leave substrate binding intact

Vmax decreases, Km stays the same

Noncompetitive inhibition lineweaver burk

slope and y intercept both increase

lines meet on x axis

methotrexate

competitive inhibitor tat resembles the folate substrate of DHFR

binds to active site where folate is suppose to bind

increased substrate concentration competes it away - v max same Km inc

needed for nucleotide synthesis

allosteric enzymes

binding at one site changes the activity at another site

on a V0 vs S plot, the curve is sigmoidal instead of hyperbolic

have T state and R state

can be homo or heterotropic

T states and R states

T-state: low affinity for substrate

R-state: high affinity for substrate

homotropic vs heterotropic effectors

homotropic - the substrate binds

heterotropic, other molecule binds

Hill coefficient

V0 = Vmax[S]n / (K0.5n + [S]n)

n = 1 hyperbolic curve, no cooperativity

n > 1 positive coop, sigmoidal

N<1 negative coop