biochem exam 2

enzymes catalyze reactions under what conditions?

mild conditions - 37 degrees C, 1 atm, PH 7, aqueous environments

where is fumerate reaction located

in the mitochondrial matrix

Rate enhancement =

RE, the amount of free energy needed to enhance rate of rxn, change in G uncat - change in G cat

If G reactants > G products…

(-) G for reaction

If G reactants < G products…

(+) G for reaction

what does rate enhancement mean in words

if the catalyst provides x amount of free energy we can enhance the rate by __ times.

Effect of change in G on RE

small changes in change in G can have a large change on the rate

with a fixed amount of enzyme why does the curve flatten off

system becomes saturated because enzyme is fixed and there is way more substrate, enzyme active site becomes saturated

What is the steady state assumption

the amount of ES does not change over the course of the reaction, allows us to set the formation of ES and the breakdown of ES equal. Important for michaelis-menten equation

for michaelis menten what part of the reaction is monitored

only the initial reaction velocity

What does Km stand for

michaelis constant - measures the stability of the ES complex - low Km = forms complex more readily (good)

What does Kcat stand for

Rate constant for conversion of ES to E + product, called the turnover number for the reaction - # of products formed per time interval (sec -1)

Most useful comparison of enzyme reactions

Kcat/Km - specificity constant

enzyme with highest affinity to substrate

lowest Km

enzyme and substrate with greatest turnover # or products formed per time interval

highest Kcat

Catalytically perfect enzyme requirements

K cat needs to be in sec-1

Km has to be in molarity (M)

Kcat/Km = 10^8 - 10^9

what is michaelis menten plot used for

determining kinetic constants from data

Substrate (x) vs velocity (y)

What is the lineweaver burk plot used for

allows visualization of trends in Km and Vmax

1/substrate (x) vs 1/velocity (y)

What does the slope represent in lineweaver plot

slope = km/vmax

what does y intercept represent in lineweaver plot

y int = 1/vmax

what does x intercept represent in lineweaver plot

x int = -1/Km

which points are most reliable in lineweaver plot

x and y int, this are near the high substrate points which are the most accurate

slope is not as accurate because it is highly affected by points to the far right that are low substrate with the highest amount of error

atorvastatin

commonly prescribed to slow biosynthesis of cholesterol in the liver

• used to treat high lipid levels in the blood and prevent cardiovascular disease

• competitive inhibitor

Reversible inhibition

• noncovalent (reversible covalent) - not fixed

• noncooperative enzyme

• inhibitor can bind to enzyme alone (E), ES complex, or both

Reversible inhibition - inhibitor binding to enzyme alone

• Ki - inhibitor dissociation constant for the EI complex

• the lower the Ki the tigher the binding between enzyme and inhibitor

Reversible inhibition - inhibitor binding to ES complex

• Ki’ - inhibitor dissociation constant for ESI complex

• lower the Ki’ the tighter the binding between ES complex and inhibitor

what should inhibitors do to Vmax

Vmax should always decrease with inhibitors

competitive inhibition - from weak to strong

1. molecules that interact with residues around or in active site

2. substrate analogs - used to understand interactions between substrate and enzyme

3. transition state and intermediate analogs - used to understand the catalytic pathway

uncompetitve inhibition

• inhibitor binds only ES or EP complex -stops turnover

• no way to minimize effect of inhibition

• higher substrate = more inhibition

Mixed inhibition

• inhibitor binding site is not part of active site - can bind both E and ES leading to mixture of competitive and noncompetitive

• binding causes conformational change, inactivates enzyme

How to recognize competitive

• v max is the same (y intercept is the same)

• Km increases - moves toward positive x axis

How to recognize uncompetitive

• parallel

• Km and vmax drop proportionally

how to recognize mixed competitive

• binds with higher affinity to E than ES

• a > a’

• V max is smaller (y int)

• Km is bigger

how to recognize mixed non competitve

• binds with equal affinity to E and ES

• a = a’

• vmax is smaller

• Km = Km control

how to recognize mixed uncompetitive

• binds with lower affinity to E than ES

• a < a’

• vmax is smaller

• Km is smaller - not proportional