Lectures 18-25, Enzymes, regulation, Citric Acid Cycle, Glycolysis + Gluconeogenesis Flashcards
(157 cards)
1
Q
What is Sir Archibald Edward Garrod known for?
A
First to make a connection between
disease and fundamental errors in
biochemical reactions
• Coined term “inborn errors of
metabolism”
• Enzymes must be link
– Thus, mutations in enzymes can cause
disease by altering biochemical reactions
2
Q
What: – Contains a unique microenvironment, usually void of water, and controls the proper
shape, pH and polarity for substrate binding and chemical reactivity
A
Active Site
3
Q
Define: Transition state
A
– Intermediate structure that is not the substrate and not yet the product
• Unstable and highest free energy
4
Q
Define: Apoenzyme
A
enzyme without its cofactor
5
Q
Define: Haloenzyme
A
Cofactor bound and catalytically active
6
Q
Coenzymes are small organic molecules often derived from _________
A
vitamins
7
Q
When coenzymes are bound tightly they are called _______
A
prosthetic groups
8
Q
Examples of cofactors
A
Metals [smallest cofactor], coenzymes (vitamins - Biotin)
Stabilize
9
Q
In alcohol dehydrogenase:
______ is reduced to ______ in the conversion of ethanol to acetaldehyde
The cofactors _____ and _____ are in a specific arrangement for this catalysis
A
Slide 27 - hint hint might be on test with a couple of questions
NAD+, NADH
NAD+, Zinc
10
Q
Which class of enzymes is associated with the this type of reaction:
Oxidation-reduction
- transfering electrons from one molecule to another
A
Oxidoreductases (ex. Lactate dehydrogenase)
ex. Alcohol dehydrogenase - converts alcohols to aldyhydes or ketones - Alcohol is oxidized and NAD+ is reduced
11
Q
Which class of enzymes is associated with the this type of reaction:
Group transfer
Donor molecule required
A
Transferases (ex. nucleoside monophosphate kinase)
Coenzyme A transfering Acytl groups
Kinase receptors - phosphorylate proteins and themselves
12
Q
Which class of enzymes is associated with the this type of reaction:
Hydrolysis reactions (transfer of functional groups to water)
Enzyme that catalyzes the hydrolysis of covalent bonds
transfer of functional groups to water
A
Hydrolases (ex. Chymotrypsin)
Ex. Disaccharidases - cleave double sugars to single sugars
Ex. defects in lactase - lactose intolerance
13
Q
Which class of enzymes is associated with the this type of reaction:
Addition or removal of groups to form double bonds
Unique active site that can transfer electrons to active site and move them around on the product. Normally one substrate becoming two products with the formation of a double bond.
A
Lyases (ex. Fumarase)
Ex. Alsolase
14
Q
Which class of enzymes is associated with the this type of reaction:
Isomerization (intramolecular group transfer)
Intramolecular oxidation-reduction reaction (group transfer)
Don’t lose any products, just move a double bond
A
Isomerases (ex. triose phosphate isomerase)
**making an isomer
15
Q
Which class of enzymes is associated with the this type of reaction:
Ligation of two substrates at the expense of ATP hydrolysis
Creating the covalent linkage of two substrates
A
Ligases (Aminoacyl-tRNA synthetase)
Ex. DNA ligase - joining two DNA molecules, two phosphodiester bonds between the two molecules.
16
Q
T/F: enzymes are biocatalysts that follow classical chemical laws. They speed up the rate at which a chemical reaction reaches equilibrium and alter the final concentrations of the reactants at equilibrium
A
False: they do not alter the final concentrations
17
Q
What is enzyme kinetics?
A
Enzyme kinetics is the study of biochemical
reactions that are catalyzed by enzymes. The
focus of which is the reaction rates of these
enzymatic reactions.
Enzymes affect reaction rates by lowering the activation energy (ΔG‡) for the reaction.
18
Q
Steady-state kinetics assumptions:
A
- ES is in rapid equilibrium with E and S
- Rate of ES formation = rate of ES breakdown
– Therefore [ES] is essentially constant - [S] >>> [E]
– Therefore [S] is essentially constant - Initial velocity of reaction is measured from
time = 0 (thus, V0)
19
Q
KM is the substrate concentration that produces
______ for the catalyzed reaction.
A
½ Vmax
• KM has units of molarity (M)
• KM is independent of enzyme amount and purity
• KM is dependent on assay conditions such as pH,
presence of inhibitors or activators, temperature,
and ionic strength. This is because that KM is
constructed purely of rate constants.
Km is called Michaelis constant
20
Q
____ is the maximum velocity - at which enzyme is
saturated with substrate.
A
Vmax
• Vmax has units of μmol/min
• Vmax is dependent on amount of enzyme used.
• Vmax is the highest reaction rate that can be
attained because all of the enzyme is
saturated with substrate.
21
Q
kcat =
A
Vmax/[Etotal]
kcat is called either the catalytic constant or turnover number
• If kcat = 1000 sec-1, the enzyme can convert 1000 molecules of substrate into product each second at saturating [S]
**number of molecules that can be converted to product per second.
22
Q
On a Lineweaver Burk Plot the y intercept =
A
1/Vmax
23
Q
On a Lineweaver-Burk Plot the x-intercept =
A
-1/Km
24
Q
On a lineweaver burk plot the slope =
A
Km/Vmax
25
A low Km means what about the affinity of the active site?
high affinity at active site.
26
What is the name of the concentration at which half the active sites are filled?
Km
27
What is the measure of catalytic efficiency?
kcat/Km
higher the kcat/Km value the more efficient te enzyme (limits - 10^9. Cannot be more efficient than standard diffusion)
28
How is the lineweaver burke plot derived?
it is the reciprocal of michaelis-menton
michaelis menton: Vo= Vmax\*[S]/(Km+[S])
lineweaver burke: 1/Vo = Km/ (Vmax\*[S]) + 1/Vmax
29
\*\* Look at tables of Lineweaver Burke, be able to get Km and Vmax, etc.
30
What type of reaction:
All substrates bind to the enzyme before any product
is released
Sequential reactions
both substrates form some product. ex. NADH binds first, then creates better binding site for pyruvate, then lactate is formed and NAD+ is released
31
What type of reaction:
One of the products is released before a second
substrate binds
Double-displacement (ping-pong) reactions
One substate binds, a product is released, then a second substrate comes in and binds and a second product is released.
32
Where is Sirt1 found in the cell?
In the nucleus
33
Where is Sirt3 found in the cell?
in the mitochondria
34
Define: Reversible Inhibition
bind to enzymes with **non-covalent interactions** such as H+ bonds, hydrophobic interactions and ionic bonds and do not undergo chemical reactions when
bound to the enzyme and **can be easily removed by dilution or dialysis**.
**Mimics the substrates**
– EXAMPLE: HIV protease inhibitors: mimics the enzymes’ substrates
35
Define: Irreversible Inhibition
```
**covalently modifies an enzyme**, and **inhibition cannot therefore be reversed**. These inhibitors aregenerally specific for one class of enzyme and do not
inactivate all proteins; they do not function by destroying
protein structure but by specifically altering the active site of
their target
```
**"kills the enzyme"**
– Example: FFR-CK (Phenlalanine-phenelalanine-arginine
chloromethylketone) covalently binds to the active site serine in
plasmin.
36
What are the Three kinds of Reversible Inhibition and what are their effects?
1. Competetive - **Increase effective Km **(More substrate needed because active site are filled by inhibitors). Inhibits at the active site.
2. Uncompetetive - Binds only to the enzyme-substrte complex. **Both Km and Vmax are altered.** DOES NOT inhibit at the active site.
3. Non-competitive - **Affects only the Vmax**. can bind with both the enzyme and enzyme substrate complex.
**LOOK AT GRAPHS FOR EACH!!!!! Be able to indentify!**
37
How does pH effect enzyme activity?
pH can denature and change effectivness (kcat)
Changes in pH may denature enzymes by
altering the enzyme's charge
altering the ionic bonds of the enzyme that
contribute to its functional shape
Enzymes have an optimal pH that helps
maintain their 3D shape
38
What two outcomes happen with Allosteric regulation?
allosteric inhibition stabilizes the enzyme in it's low affinity form resulting in little or no activity
**OR**
stabilizes it in high affinity form, resulting in enzyme activity
39
Review slide 30
ATCase is what type of enzyme modification?
Transferase
condensation reaction joining two molecules with loss of a small molecule
40
ATCase is found in what two distinct conformational states?
textbook example of allosteric modification (products inhibit upstream reactions)
T state (less active - **favored by CTP binding - inhibits the enzyme**) and R state (more active - relaxed/open - ** Favored by substrate binding**)
**Zinc is a cofactor in ATCase**
41
Why do you have the multiphasic curve in the formation of N-carbamoylaspartate (ATCase)?
Combination of T and R state curves. - Sigmoidal curve
42
Serine proteases use what for the active site?
**Use active site serine for peptide cleavage**
43
Cleavage of peptide backbone in serine proteases occurs in which step?
3rd.
Go over slide 52
Acyl-anzyme intermediate created via the serine
44
Where is Pepsin synthesized?
Stomach
45
Where is Chymotrypsin, Trypsin, Carboxypeptidase, and Elastase synthesized?
Pancrease
46
Anabolic are:
those that require inputs of energy to proceed
47
\_\_\_\_ Is the Universal Currency of Free Energy in
Biological Systems
ATP
48
In aerobic organisms, the ultimate electron acceptor in the
oxidation of carbon is \_\_\_\_
and the oxidation product is \_\_\_\_\_
.
O2, CO2
49
Which of the following has the most energy released during oxidation?
A. Methane
B. Methanol
C. Formaldehyde
D. Formic Acid
E. Carbon Dioxide
A. Methane
A-\>E is most to least amount of energy, Slide 10
50
Oxidation of 2-carbon units to produce
2 CO2 molecules
1 GTP
Electrons in the form of NADH and FADH2
51
3 ways metabolic processes are regulated:
1) Controlling the amounts of enzymes
Transcription/signaling
2) Controlling catalytic activity
Feedback inhibition – conformation/modification
3) Controlling the accessibility of substrates
Compartmentalization of pathway
52
Citric acid cycle general characteristics:
Take place in mitochondria matrix, etc...
53
Under anaerobic conditions pyruvate
is converted to \_\_\_\_\_\_\_\_\_\_
lactate or ethanol
54
State the Prosthetic group, Reaction catalyzed, and # of chains:
1. E1
2. E2
3. E3
1. 24 chains, TPP, Oxidative decarboxylation of pyruvate
2. 24 chains, Lipoamide, Transfer of acetyl group to CoA
3. 12 chains, FAD, Regeneration of the oxidized form of lipoamide
Slide 28 table
55
First step in Citric Acid cycle is what?
Generation of Acetyl CoA
56
Pyruvate is fromed via what process?
Glycolysis
57
Draw out and go over slide 39!!
58
T/F: Prosthetic groups are modified and put back into the origional form for each step of the reaction?
True
59
How can pyruvate dehydrogenase be regulated?
Allosterically and by reverse phosphorylation
Slide 47
high Acetyl CoA inhibits E2
Products also increase phosphorylation of PDH E2
Accumulation of ADP and pyruvate activate phosphatases
60
The citric acid cycle occurs under ______ conditions and
produces _____ energy from glucose than glycolysis
aerobic, more
61
What is the function of PDH?
```
Pyruvate dehydrogenase (PDH) links glycolysis to the
citric acid cycle
```
62
PDH contains ___ enzymes and uses ____ cofactors to
generate acetyl CoA for entry into the citric acid cycle
3, 5
63
The Citric acid cycle starts with what 2 carbon molecule?
Acytl CoA
64
The first step of the TCA is to generate:
and the enzyme used is:
and what is condensed?
Citric Acid (Citrate)
Citrate synthase
4-carbon oxaloacetate is condensed with 2-carbon of acetyl CoA
65
Which binds first Ozaloacetate or Acetyl CoA?
Oxaloacetate, **wont bind unless enough oxaloacetate is around**
66
T/F: The first step of TCA cycle exhibits sequential, ordered kinetics
True
67
The second step of the TCA uses what enzyme/substrate, and produces what?
Citrate (from the end of the 1st step)
Aconitase is the enzyme (contains iron sulfur complex)
Isocitrate is produced
68
\*\* First major regulatory step: 3rd step:
What substrate, enzyme, product
First of 4 redox reactions
Isocitrate
Isocitrate dehydrogenase
a-Ketoglutarate
**loss of electrons in form of NADH**
69
What is the rate limiting step of the TCA?
3rd step
That 5 carbon's best path is to remain in the cycle, isocitrate could go back to acetyl CoA if energy is not needed
70
What allosterically regulates the 3rd step of TCA?
ADP
71
a-ketoglutarate dehydrogenase complex is very similar to what?
pyruvate dehydrogenase
72
4th step of TCA substrate, enzyme, product
a-Ketoglutarate,
a-Ketoglutarate **dehydrogenase** complex,
succinyl CoA
73
5th step of TCA, substrate, enzyme, product
Succinyl CoA
Succinyl CoA synthetase
Succinate
only step that directly yeilds **GTP formed**
74
6th step of TCA substrate, enzyme, product
Succinate
Succinate **dehydrogenase**
Fumarate
**FADH2 formed**
75
7th step of TCA substrate, enzyme, product
Fumarate
Fumarase
Malate
76
8th step of TCA substrate, enzyme, product
Malate
Malate **dehydrogenase**
Oxaloacetate!
77
Which reaction of the TCA yields GTP?
Succinyl CoA synthase
78
Which amino acid transfers the phosphate group in the succinyl CoA synthase reaction?
Histidine
79
How many redox reactions are there from Succinate to Oxaloacetate?
2 (final state of cycle), 2 steps of oxidation
80
electrons from FADH2 are transferred directly to what?
Coenzyme Q of electron transport chain,
16 mins into lecture
81
\_\_\_\_\_\_\_\_\_\_\_\_\_\_ catalyzes formation of fumarate
while generating FADH2
Succinate dehydrogenase
82
If Acetyl CoA is not needed for energy it is transformed into:
Lipids
83
We have a high level of Acetyl CoA, is PDH on or off?
On
84
Low energy charge means we have a lot of ADP or little ADP
a lot
85
review slide 81!!!
86
Accumulation of NAD means what?
we need more energy
drives the reaction (a substrate for PDH)
87
Phophatases can also be stimulated by what ion to increase muscle contraction?
Ca2+
88
T/F: Insulin can stimulate fatty acid synthesis by activating phosphatases
True
slide 83
89
α-ketogluturate dehydrogenase is Allosterically inhibited by its products,
\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_
succinyl CoA and NADH
90
α-ketoglutarate that builds up
from enzyme inhibition can be used
for synthesis of \_\_\_\_\_\_\_\_\_\_\_
amino acids and purine bases
91
Oxaloactetate is
replenished by \_\_\_\_\_\_\_\_
pyruvate
92
\_\_\_\_\_\_\_\_\_\_\_\_ are a source for citric acid cycle
intermediates
Branch chain amino acids
slide 89
93
definition: Anaplerotic reactions
Using our building blocks to create energy (intermediates)
**we have low energy**, not enough substrates to create intermediates, ATP production is sustained.
94
Which is faster, anaerobic ATP generation or aerobic ATP generation
anaerobic
95
Glycolysis is..
Sequence of reactions that metabolizes one molecule of glucose to two molecules of pyruvate and generates the net production of two molecules of ATP
1. This is an anaerobic process (no O2)
2. Pyruvate can be completely oxidized under aerobic conditions, generating much more ATP
96
What occurs in the Hexokinase step of glycolysis?
Formation of glucose 6-phosphate
Phosphorylation traps glucose in cell
Irreversible step with hexokinase
97
What occurs in the Phosphoglucose
step of glycolysis?
Isomerization of glucose 6-phosphate to fructose 6-phosphate (F6P) (from an aldose to a ketose)
**Readily reversible **
**Phosphoglucose isomerase used**
98
What occurs in the Phosphofructokinase step of glycolysis?
1) The "commited step", reversible up to this point
2) second phosphorylation, formation of formation of fructose 1,6-bisphosphate (F-1,6-BP)
**Phosphofructokinase is a key enzyme in the regulation and integration of much of metabolism**
99
Stage 2: Aldose step, involves?
Cleavage of 6-carbon F-1,6-BP into two 3-carbon phosphate compounds
readily reversible
100
Stage 2: Triose phosphate isomerase step of glycolysis involves?
Interconversion of DHAP and **G3P**
Equilibrium is 96% DHAP and 4% G3P, but metabolism of G3P in remaining steps of glycolysis pulls reaction toward G3P
c. Reaction is reversible
101
Which of the following enzymes catalyzes the committed step in glycolysis?
## Footnote
A. Glucokinase
B. Hexokinase
C. Phosphofructokinase 1
D. Phosphofructokinase 2
E. Pyruvate kinase
C. Phosphofructokinase 1
102
•What is the difference between aerobic and anaerobic glycolysis?
103
•What is the difference between allosteric and covalent regulation?
104
•What is the role of fructose 2,6-bisphosphate in regulating glycolysis?
105
•Why is glucose 6-phosphate “trapped” in most cells?
it is phosphorylated
106
•What are the “by-pass” reactions of gluconeogenesis?
107
•What is the role of the malate shuttle in gluconeogenesis?
108
•How is glucose 6-phosphate dephosphorylated?
109
•How are glycolysis and gluconeogenesis reciprocally regulated?
110
Which of these enzymes is used in gluconeogenesis to by-pass one of the irreversible steps in glycolysis?
A. Glucokinase
B. Glyceraldehyde 3-phosphate dehydrogenase
C. Pyruvate carboxylase
D. Pyruvate kinase
E. Phosphofructokinase 2
C. Pyruvate carboxylase
111
What three reactions in glycolysis are irreversible?
Hexokinase
Phosphofructokinase
Pyrovate kinase step
112
In the liver, during gluconeogenesis, what enzyme is used instead of hexokinase?
Glucose 6-phosphatase
113
In the liver, during gluconeogenesis, what enzyme is used instead of phosphofructokinase?
Fructose 1,6-biphosphatase
114
In the liver, during glycolysis, what enzyme is used instead of hexokinase?
glucokinase
115
Gluconeogenesis uses all of the following EXCEPT:
A. Lactate
B. Fatty Acids
C. Amino Acids
D. Glycerol
B. Fatty Acids
116
Gluconeogenesis converts ______ into \_\_\_\_\_\_\_\_
pyruvate, glucose
117
T/F: Gluconeogenesis is the reversal of glycolysis
F.
118
Which 3 steps in gluconeogenesis require ATP/GTP?
pyruvate-\>oxaloacetate
Oxaloacetate-\> phosphoenolpyruvate
3-phosphoglycerate-\> 1,3-Bisphosphoglucerate
119
About how many ATP does it take to convert 2 pyruvates to 1 glucose molecule?
6 ATP (3 for each pyruvate, gluconeogenesis has 3 steps that require ATP input)
120
In gluconeogenesis, Oxaloacetate is transported from the mitochondria to the cytoplasm via:
malate shuttle
Oxaloacetate is actually converted to Malate, transferred across the membrane, and then back to Oxaloacetate
121
Where in the liver cell is glucose-6-phosphatase (almost like a membrane protein) located?
ER lumen
SO
When you dephosphorlyate G6P in liver cells, this happens in the ER. The glucose is then transported out of the ER.
122
What determines whether gluconeogenesis or glycolysis will be most active?
Energy charge (ATP/ADP ratio)
123
When ATP levels are high, would you get glycolysis or gluconeogenesis in the liver?
gluconeogenesis
124
What is the shuttle for pyruvate into the liver?
Alanine
used when muscle cells metabolize amino acids for fuel and have pyruvate as a by product. This pyruvate is then transported as Alanine to the liver.
125
Fructose 2,6 bisphosphate activates _______ and inhibits \_\_\_\_\_\_\_
phosphofructokinase, fuctose 1,6 biphosphatase
126
Glucagon stimulates ____ when blood glucose is rare. This causes an ______ of gluconeogenesis and a _____ of glycolysis
Protein kinase A, stimulation, inhibition
127
At high blood glucose levels, High levels of fructose 6-phosphate stimulate \_\_\_\_\_\_, which in turn ______ glycolysis and _______ gluconeogenesis.
phosphoprotein phosphatase, stimulates, inhibits
128
The Cori cycle, involves what?
converting lactate that was produced in skeletal muscle and RBCs back to pyruvate in the liver
129
Glycogen is stored in liver, muscle, and other tissues as \_\_\_\_\_\_\_
granules
granules comprised of glycogen and enzymes required for metabolism.
130
Glycogen degradation is also called
glycogenolysis
131
Glucose phosphorylase continues to break down glycogen into glucose 1-phosphate until it gets to ___ residues from the branch point.
4
132
When is vitamin B6 used as a cofactor?
Glycogen phosphorylase step
133
What is the enzyme that transfers glucose from the branch point to the core chain?
Transferase
134
what enzyme breaks the branch point, (named for the name of the link)
alpha-1,6 glucosidase
135
What enzyme converts glucose 1 phosphate to glucose 6 phosphate?
Phosphoglucomutase
136
What does branching of glycogen do?
increases solubility, increases rate at which glycogen can be synthesized and degraded.
137
branching enzyme breaks _____ bonds and forms _____ bonds by transfering ____ glucoses
Alpha 1,4
Alpha 1,6
7-8
138
The primer of glycogen is formed using glucose residues and what enzyme?
glycogenin
139
What enzyme forms alpha 1,4 bonds onto existing glycogen chains
glycogen synthase
140
Which enzyme combines UTP and glucose 1-phosphate to form UDP-glucose?
UDP-glucose pyrophosphorylase step
141
phosphorylation of glycogen phosphorylase increase or decreases activity?
increases activity
142
phosphorylation of glycogen synthase increases or decreases activity?
decrease
143
Insulin triggers phorphorylation or dephosphorylation in liver and muscle tissue?
dephosphorylation
144
Epinephrine triggers phosphorylation or dephosphorylation?
phosphorylation
145
Glucagon triggers _______ in the liver
phosphorylation in the liver
146
What is signal transduction?
the formation/release of a second messenger in response to binding of hormones (first messenger)
147
cAMP is promoted by:\_\_\_\_\_\_
cAMP activates:\_\_\_\_\_\_\_\_ which activates \_\_\_\_\_\_\_\_\_\_
Epinephrine and glucagon.
Protein kinase A
Phosphorylase kinase and glycogen phosphorylase
**EXAMPLE OF BIOCHEMICAL AMPLIFICATION**
148
Exercise:
cAMP (increases or decreases)
Protein Kinase A (active/inactive)
Phosphorylase kinase (active/inactive)
glycogen synthase (active -a/inactive - b)
phorphorylase (active-a/inactive b)
cAMP: increases
PKA - active
phorphorylase kinase - active
glycogen synthase - inactive
phorphorylase - active
149
Insulin activates \_\_\_\_\_\_
Phosphoprotein phosphatase 1
**which, dephosphorylates**
1) glycogen synthase (activates)
2) phosphorylase kinase (deactivting phosphorylase a)
3) glycogen phosphorylase (deactivate)
150
Phosphorylase kinase is activated by what ion in skeletal muscle?
Calcium - which breaks down more glycogen to provide a inflow of energy
151
IN THE LIVER, high levels of glucose 6 phosphate activate ______ even when it is phosphorylated
glycogen synthase
152
IN MUSCLE, glycogen phorphorylase is activated by \_\_\_ and inhibited ATP and G6P
AMP
153
Blood glucose levels are maintained primarily through the storage, synthesis, and release of glucose by \_\_\_\_\_\_\_
the liver
154
What organ releases both insulin and glucagon?
pancreas
155
Which of the following directly activates glycogen phosphorylase?
A. Adenylate cyclase
B. cAMP
C. Protein kinase
D. Phosphorylase kinase
E. Phosphoprotein phosphatase
D. Phosphorylase kinase
156
Does insulin or glucagon take part in allosteric regulation?
No, signal transduction
157
