Biochemistry Exam 4

Benefits of enzymes

• Higher reaction rates

• Mild conditions

• Greater specificity

Enzymes rarely produce side products

true

Enzymes are composed of

proteins

Metal atom bonded to a protein

cofactor

Holoenzyme

protein + cofactor

Apoenzyme

protein without REQUIRED cofactor

Active site

Where substrate binds

The active site creates a unique

microenviornment

Reaction rate

collision frequency X energy factor X probability factor(oreintation)

how do enzymes enhance the probability factor

by oreinting the substrate in an optimal postion

How do enzymes lower the energy factor

• Stabilizes the transition state of substrate

• replaced interaction with water in solvetaed state

• directs the path leading to only one product formation

How do enzymes increase the collsion factor

Reactants bound at an enzyme are at infintite conentration

Change of G = ____ at equilibrium

0

Negative change of G reflects

a spontaneous reaction

Change of G relates to

Rate of Reaction

An enzyme cannot

alter the postion of the equilibrium

A enzyme only allows the reaction to

Reach equilibrium faster

An anzyme increases the rates of the

forward and reverse reactions equally

Binding energy

facilitates the binding of E+S into the ES complex

2 parts of active site

1. specificity

2. Catalytic

What will happen if an enzyme has too much specificity

the ES complex will become too stable and not go into the transition state

To keep the enzyme from getting stuck at the ES phase, enzymes must be complementary to

Transtion state NOT the substrate

2 ways active site can bind

1. Lock and Key —>affinity for substrate without conformation change

2. Induced fit —> Site increases affinity for the substate with a conformational change upon binding

Maximum velocity is reached when

all the active binding sites are filled with substrate

Reaction rate is limited by two things

1. How many active sites are avalible

2. How fast the enzyme works

In a higher the concentration the rate __________

increases (until all the active sites are taken up and the speed flatlines)

V

number of moles of product formed per second

Michaelies-Menten is a plot of

V vs [S]

Michealies-Menten shows a _____ relationship at low [S] concentrations

linear

At high [S] in they enhance the probaility factor we close in on _____ ebing the only thing in solution

ES complex

Velocity of a reaction =

rate constant of step X [reactants]

Steady state

the concentration of intermediates remains constant over time because their rate of production equals their rate of consumption.

[E] =

[total enzyme] - [ES] (complexing enzyme)

Michealis-menten equation if [S] is large

V₀ = V_max (([S]) / ([S}+Km))

Km

(the Michaelis constant) is defined as the substrate concentration required to reach half the maximum velocity ()

Lineweaver-Burk Plot

y intercept of Lineweaver-burk gives

1/Vmax

x intercept of lineweaver Burk plot

-1/Km

Slope of linearweaver Burk Plot

Km/Vmax

y of lineweaver burk

1/V0

x of lineweaver burk

1/[S]

Eadie-Hofstee plots

V₀ vs V₀/[S]

Eadie-Hofstee Y intercept

V_max

Eadie-Hofstee slope

-Km

Km = [S] when

hald of the active sites on the enzyme are filled

Small Km

high affinity of enzyme for subtrate (k1 is high)(fomration of ES complex is high)

large Km

low affinity of enzyme for subtrate (K2 is high)( decompostion of ES complex is high)

turnover number

number of substrate molecules converted into products by an enzyme per unit of time when FULLY saturarted with substrate

K_cat (turnover number) is equal to

K₂

Vmax

represents the maximum rate (velocity) an enzyme-catalyzed reaction reaches when the enzyme is fully saturated with substrate

[E_t]

number of active sites

Vmax = ________

k₂ [E_t]

Limit to Vmax

a reaction cannot proceed faster then the substrate can find the enzyme (rate of diffusion)

Two types of enzyme inhibition

• Irreversible inhibition —> stop enzyme and it dies forever

• reversible inhibition (4 types) —> remove the drug and remove the inhibition

Irreversible Inhibitor (I) will bind _______ to enzyme at active sight

TIGHTLY —> covalently links

Most Irreversible Inhibitors (I) are often

alkylating agents that react with active site of AA residues

Competivtive Inhibition

I binds to the same active site as the substrate.

Competivtive Inhibition can be overcome by

high [S]

How does Vmax and Km change with competitive inhibtion

• Km​ increases → the enzyme appears to have lower affinity for the substrate. It takes a higher substrate concentration to reach half of Vmax​.

• Vmax​ unchanged → if you add enough substrate, the substrate can outcompete the inhibitor and the enzyme can still reach the same maximum rate.

α represents

degree of Inhibition

A larger α means

more inhibition

α =

slope + inhibitor / slope uninhibited enzyme

K_i

Inhibitor dissocitation constant —> How well the inhibior biunds to the enzyme

Low K_i

inhibitor binds tightly to enzyme —> less concentration is needed to achieve 50% inhibition

high K_i

inhibitor binds loosley to enzyme

K_i =

[I] / (α-1)

Noncompetitive Inhibition

S and I bind at the same time

I will inhibit by decreasing the turnover number

Cannot be overcome by an increase in [S]

Noncompetitive Inhibition effects on Vmax and Km

• Vmax decreases —> the inhibitor decreases full activity

• Km is not altered —> The inhibitor does not change substrate binding affinity, it takes the same [S] to reach 1/2 new vmax

mixed inhibition

Both Vmax (decrease) and Km change (K could increase or decrease)

Uncompetitive Inhibition

Inhibitor can only bind to ES compelx

Both Km and Vmax are decreased

Competitive Inhibition

Noncompetitive Inhibtion

Mixed Inhibition

Uncompetitive Inhibtion

Serine Protease

Class of enzymes with a reactive serine OH at their active site

Serine Protease enzymes are

digestive enzymes that catalyze the hydrolyssi of peptide bonds

Chymotrpsin, trypsin and eleastase are all

Serine Protease digestive enzymes

Protease

Breakage of peptide bonds

What class of protease is chymotrypsin?

Serine

Chymotrypsn in vivo specifically hydrolyzes

• peptide bonds on the carboxyl side of aromatic bulky side chains

• esters

Bulk aromatic amino acids

Phenylalanine (Phe), Tyrosine (Tyr), and Tryptophan (Trp)

How many amino acids are in the active form of chymotrypsin

241

Overall rate determining step

Slow step (usually after the burst phase)

Burst phase

Inital reaction phase were a lot of product is formed quickly

Chymotrypsin specficity

bulky nonpolar

Chymotrypsin binding pocket

Large and nonpolar

Trypsin specificity

Arg and Lys (+) residues

Trypsin binding pocket

Negatively charged residues

Elastase specificty

Small nonpolar

Elastase binding pocket

Thr and val residues close off binding pocket so that only small residues can be accommodated

Catalytic triad

Serine, Histidine, Aspartic Acid

Enolase

catalzyses a step of glycosis

Enolase Co-factor

Mg

Enolase facilates a

dehydration

Ribonuclease A is secreted by the

pancreas

Ribonuclease A catalzyes the

hydrolysis of phosphodiester bonds in RNA