Download LECT24 enz2

Enzyme Dialogue
How to decipher the meaning of
enzyme terms
Enzyme Activity
Definition, Meaning and
Measurement of ...
What is an Enzyme?
An enzyme is a protein that behaves as a catalyst
How do we know how much enzyme is present in a
cell, a tissue, an extract, etc.?
We measure the amount of enzyme by measuring
its activity.
Come Again!
An enzyme is measured by what it does not by what
it is.
Why is it done that way?
Because most of the time we never have a pure enzyme.
Therefore, we measure its amount indirectly by its activity
What do you mean by activity?
Activity is a velocity. We measure the rate of change of
of a specific amount of substrate per unit time.
What are the conditions?
We must assume the enzyme is limiting. Therefore, the
substrate must be in abundant supply.
How do you turn activity into enzyme?
We do this by assigning a unit of activity and relate that
to the enzyme.
What is a Unit?
A unit is the amount of enzyme that catalyzes the
change of one micromole of substrate in one minute.
How does that tell me how much enzyme is present?
If we relate enzyme units to micrograms of enzyme, we can
immediately determine the weight of enzyme by knowing
the activity.
What if the enzyme is not pure?
We then relate activity to mg of protein present. That
measurement gives us the specific activity.
What if the enzyme is pure?
Then we can express activity as a turnover number.
What’s that?
Turnover number is a time-dependent change expressed
as moles of substrate changed per mole of enzyme present.
Unit of Enzyme Activity
Amount of enzyme sufficient to convert one
micromole of substrate to product in one minute
Example
An extract catalyzed the change of 20 micromoles
of substrate to product in 5 minutes. How many
units of enzyme was present in the extract?
ANSWER
4 UNITS OF ENZYME
Specific Activity
Units per mg of protein
Example
5 mg of protein were in an extract that
catalyzed the change of 100 micromoles of
substrate to product in 10 minutes. What
is the specific activity of the enzyme?
Answer:
2 units per mg of protein
Practical Meaning of Specific Activity
Relates enzyme activity to protein.
Gauges purity of an enzyme
E
P
E
E
E
P
E
P
E
P
E
=
E+P
6
10
E
P P
E
P P
P
E
P
E
E
E+P
=
4
10
Specific Activity and Protein Purification
Table 1. Purification of Lactic acid dehydrogenase from pigeon liver
Preparation
Activity
(mole sub/min)
1. Crude extract
Protein
(mg)
Specific Activity
(mole sub/min/mg protein)
1,565
7,500
0.21
2. (NH4)2SO4 cut
528
420
1.26
(6-fold purer)
3. DEAE cellulose
450
52
8.65
(41.2-fold purer)
4. Sephadex G-75
395
6.3
62.7
(298-fold purer)
5. Affinity Chromatography
275
3.8
72.4
(344-fold purer
Turnover Number
Relates moles of substrate changes to moles
of enzyme Present
1. Must have pure enzyme
2. Must know Molecular weight of enzyme
T.O. is synonymous with Kcat,
the catalytic constant.
Enzyme Specificity
+ NH
3
deamination
(oxidase)
R C COO
O
+
NH3
R C COO
H
R' C COO
decarboxylation
(decarboxylase)
H
+
R C NH3 + CO2
H
O
amino group transfer
(aminotransferase)
+ NH
3
R' C COO
H
+
R C COO
O
Factors that Effect Enzyme Activity
1. Temperature
2. pH
3. cofactors and coenzymes
Sharp
drop
4. Enzyme concentration
5. Substrate concentration
6. Inhibitors and inactivators
Heat
denaturation
Trypsin
Pepsin
Activity
0 10 20 30 40 50 60
oC
0 1 2 3 4 5 6 7 8 9
pH
COFACTORS AND COENZYMES
Organic
Organic Cofactor
Vitamin
Function
Enzyme Class
NAD, NADP
FAD
Thiamin pyrophosphate
Pyridoxal-5’-phosphate
Biocytin
Tetrahydrofolates
Coenzyme A
Lipoic acid
Coenzyme Q
Cobalamin
Carnitine
Niacin
Riboflavin (B2)
Thiamin (B1)
Pyridoxine (B6)
Biotin
Folic acid
Pantothene
Lipoic acid
Ubiquinone
Vitamin B12
Carnitine
electron carrier
electron carrier
group transfer
group transfer
CO2 transfer
group transfer
group transfer
group transfer
electron transfer
group transfer
group transfer
oxidoreductases
oxidoreductases
oxidoreductases
aminotransferases
carboxylases
one-carbon transfer
Synthases
oxidoreductases
electron transport
mutases, methylases
acyl group transport
Inorganic Cofactors
Inorganic Cofactor
Function
Enzyme Class
Magnesium
Calcium
Potassium
substrate binding
substrate activation
structure stabilization
kinases
hydrolases
pyruvate kinase
Iron
Zinc
Copper
Manganese
Cobalt
Selenium
oxygen binding, electron transport
substrate binding, structure stability
dioxygen activation
dioxygen activation
group transfer
peroxidation
cytochromes
DNA binding
oxidases
oxidases
mutases (with B12)
peroxidases
REDOX COFACTORS- NAD+, NADP
O
NH2
C
N
N
O
N
N
O
HO
NH2
Niacinamide
(niacin)
O
N+
CH2O-P-O-P-OCH2
O O
O
OH
O
HO
OH
HO-P-O
O
Nicotine Adenine Dinucleotide (NAD+)
Nicotine Adenine Dinucleotide phosphate (NADP+)
Active site
H
H
..
H
N+
CONH2
H
+
Reduced Coenzyme
NADH + H+
REDOX COFACTORS FAD
Pseudo glycosidic bond
OHOHOH
CH2 C C C CH2 PP ribose adenine
H HH
CH3
CH3
N
H
N
..
N
H
O
NH
Ring holds 2 electrons
and 2 protons
O
Flavine adenine dinucleotide
FAD
FADH2