Test 2 - Enzymes Flashcards
(105 cards)
1
Q
Suffix that refers to enzymes?
A
-ase
2
Q
What type of reaction is catalyzed by Oxidoreductases?
A
Transfer of electrons (Hydride ions or H atoms)
3
Q
What type of reaction is catalyzed by Transferases?
A
Group transfer reactions
4
Q
What type of reaction is catalyzed by Hydrolases?
A
Hydrolysis reactions (transfer of functional groups to water).
5
Q
What type of reaction is catalyzed by Lyases?
A
Cleavage of C-C, C-O, C-N, or other bonds by elimination, leaving double bonds or rings, or addition of groups to double bonds.
6
Q
What type of reaction is catalyzed by Isomerases?
A
Transfer of groups within molecules to yield isomeric forms.
7
Q
What type of reaction is catalyzed by Ligases?
A
Formation of C-C, C-S, C-O, and C-N bonds by condensation reactions coupled to cleavage of ATP or similar cofactor.
8
Q
What enzyme catalyzes the transfer of a phosphoryl group from ATP to glucose?
A
D-hexose 6-phosphotransferase
9
Q
Definition of Apoenzyme:
A
Inactive enzyme without its factor
10
Q
Definition of Holoenzyme?
A
Complete active enzyme with the factor.
11
Q
Define Prosthetic Groups
A
Tightly and stably incorporated into a protein’s structure by covalent or noncovalent forces.
12
Q
Define Cofactors, examples
A
Bind in a transient, dissociable manner either to the enzyme or to a substrate such as ATP.
Example: Metal Ions
13
Q
Universal Electron Carriers
A
FAD and FMN
14
Q
FAD states
A
FAD
FADH+
FADH2 (Fully reduced)
15
Q
Universal Mobile electron carriers?
A
NAD+ and NADP+
16
Q
What causes pellagra?
A
Niacin deficiancy
17
Q
Why enzymes over inorganic catalysts?
A
Work at physiological pH and temp.
High reaction rate and capaciy for regulation
High specificty for substrates at the active site.
18
Q
Two models of enzyme-substrate interactions that have been proposed?
A
Lock and key
Induced fit
19
Q
Describe the Lock and Key model:
A
High specificity due to complementary nature of binding site and the ligand.
Complementary in: Size, Shape, Charge, Hydrophobic/Hydrophilic character (SSCHH).
20
Q
Describe the Induced Fit Model
A
Conformational changes may occur upon ligand binding.
This adaptation is called the induced fit.
Both the ligand and the protein can change their conformations.
21
Q
Mechanisms of Enzyme Catalysis
A
Catalysis by Proxmity
Acid-base Catalysis
Catalysis by Strain
Covalent Catalysis
22
Q
Example of Covalent Catalysis compounds.
A
Serine protease, chymotrypsin.
23
Q
What is Enzyme Kinetics?
A
Kinetics is the sutdy of the rate at which compounds react.
24
Q
What affects the rate of enzymatic reaction?
A
Enzyme, substrate, effectors, temperature.
25
Explain Catalysis by Proximity
Effective molarity (concentration) and orientation of substrate molecules in the active site of enzymes will enhance the rate of reactions.
Substrate channeling by multi-enzyme complexes.
26
Explain Acid-Base Catalysis
Ionizable functional groups of amino acid side chains and prosthetic groups contribute to catalysis by acting as acids or bases.
27
Catalysis by Strain
Enzymes bind their substrates in an unfavorable conformation to weaken the bond that will undergo cleavage.
Example: Stickase model
28
Explain Covalent Catalysis
Formation of a covalent bond between the enzyme and one or more substrates to create a more reactive enzyme.
Example: Group transfer reactions
29
Catalysis by Fructose-2,6-Bisphosphatase role and mechanism?
Catalyzes the hydrolytic release of the phosphate on carbon 2 of fructose-2,6-bisphosphate.
Positively charged Lys and Arg residues stabilize the negative charge of the substrate.
30
Enzymes use the binding energy of substrates to organize the reactants into what?
Stable transition state (TA).
31
How is the Stable Transition State (TA) formed?
Positioning of acid-base groups to transfer protons to or from the developing transition state intermediate.
Imposing steric strain on substrates so that their geometry approaches to that of the transition state.
32
Do enzymes affect Keq?
No they do not.
33
Equilibrium constant (Keq) of an enzymatic reaction?
Keq = [P][E] / [S][E]
Keq = [P] / [S]
34
Effect of Enzymes on equilibrium?
They cannot change the equilibrium constant, but they can speed the onset to equilibrium.
35
Zero order kinetics definition
Rate of reaction is independent of substrate concentration [S], but proportional to enzyme concentration [E].
36
First order kinetics definition
Rate is proportional to concentration of substrate.
S -\> P
v = k [S]
k is a rate constant.
37
Second order reaction definition
Rate proportional to concentration of 2 substrates.
S1 + S2 -\> P
v = k [S1] [S2}
38
Pseudo First order reaction
It is a second order reaction, but [S2] is very high and [S1] is very low causing the rate to appear to depend on concentration of only one substrate.
v = k [S1]
Rate is proportional to [S1} only, since S1 is the rate limiting substrate.
39
What are the assumptions of the Michaelis - Menten Equation?
Assumes the formation of an enzyme - substrate complex (ES)
Enzymatic reactions show saturation with their substrates [S].
ES complex is in rapid equilibrium with free enzyme [E]
Breakdown of ES to form products is assumed to be slower than the formation of ES and breakdown of ES to E and S.
40
Michaelis - Menten Equation
v = Vmax [S] / Km + [S]
41
What is Km in Enzyme Kinetics
Km is the substrate concentration that corresponds to half the Vmax and it can be roughly estimated from initial velocity (v) vs [S] plot.
42
Define First order kinetics
V0 to Km
Collision of substrate with enzyme is rate limiting due to low [S].
43
Define Zero Order kinetics
The enzyme is saturated with high [S] and the rate-limiting step is the product release from the enzyme surface.
Plateau on graph.
44
Lineweaver - Burk Equation
1 / V0 = Km / Vmax \* 1 / [S] + 1 / Vmax
45
Slope of the Lineweaver - Burk plot?
Km / Vmax
46
Y-intercept of the Lineweaver - Burk plot?
1 / Vmax
47
X - Intercept of the Lineweaver - Burk plot?
- 1 / Km
48
What does a small Km mean?
Tight Binding of substrates to the enzyme.
49
What does a large Km mean?
Reflects a low affinity of the enzyme for the substrate.
50
Can enzymes have different affinities for different substrates?
True
51
When comparing efficiency of an enzyme to another, the one witht the highest k2 is?
Most effiecient at saturated [S].
52
In living cells, [S] is usually ____ and so the rate is \_\_\_\_?
<<
53
For M - M enzymes k2 = kcat what is kcat?
Catalytic rate constant or **turnover rate**
54
If the enzyme has a high K2 / Km (or Kcat / Km ) ratio what can be said?
The enzyme has reached the kinetic perfection.
55
Definition of Kcat / Km ?
The **specificity constant**, indicitive of how good an enzyme is for a given substrate.
56
What limits the maximum value for the specificity constant?
Diffusion from the active site.
57
What is the Turnover number?
Kcat
It is the number of substrate molecules converted to product per enzyme molecule per unit of time.
It is a measure of the Enzyme's maximal catalytic activity
Kcat = Vmax / Et
58
What effect does pH have on enzyme activity?
Activity of most enzymes varies with pH, most have an optimum.
The effect depends on the acid-base behavior (protonation state) of amino acids in the active site.
Optimum normally reflects physiological condition. (Except pepsin pH 1 or 2)
Extreme pH levels denature preventing substrate binding.
59
Effect of temperature on Enzyme activity
Activity increases as Temperature increases.
Usually doubles with 10ºC rise (Q10 = 2)
Most enzymes enzymes fully denatured at 70ºC
60
What is the optimum temperature for most enzymes?
30ºC
61
Definition of Single - displacement reactions and alternative name.
AKA Sequential reactions
Both substrates must combine with the enzyme to form a **ternary complex** before catalysis can proceed.
62
Define Double Displacement reaction and its alias?
Aka, Ping-Pong Mechanisms
One or more **products are released** from the enzyme before all the substrates have been added.
63
Single - Displacement Ordered Bi - Bi
E + S1 -\> ES1 + S2 -\> ES1S2 - EP1P2 -\> EP2 + P1 -\> E + P2
64
In steady - state kinetic analysis of bisubstrate reactions, ___ is varied while ___ is held constant.
And what do intersecting lines indicate?
[S1] is varied
[S2] is held constant
Intersecting lines indicate that a ternary complex is formed in the reactions.
65
Parallel lines indicate which mechanism of bisubstrate reaction?
A ping-pong (double - displacement) mechanism.
66
What are enzyme inhibitors?
Inhibitors are chemicals that reduce the rate of enzymatic reactions, they block the enzyme but they do not usually destroy it.
67
What info can an inhibitor study provide?
Enzyme mechanism
Substrate specificity
Active site aa's
Mechanism of drug action
68
What are the three types of Reversible inhibitors?
Competitive, noncompetitive (mixed), and uncompetitive inhibitors.
69
What are Irreversible inhibitors?
Covelent, permenant modification of part of enzyme required for catalysis (suicide substrates or inhibitors such as penicillin)
These types of inhibitors yield information about functional groups.
70
Competitive Inhibitor mechanism?
Inhibitor combines with free enzyme at active site (Competes with S).
I binds to the same site in the enzyme as the S, directly competing with S.
Changes the Km, but the Vmax stays the same.
71
Mixed or Noncompetitive Inhibition Mechanism?
Inhibitor (I) binds to enzyme at a site other than the active site.
This decreases the maximum velocity but does not affect Km.
Inhibitor removes a certain fractiion of the enzyme from operation regardless of the concentration of the substrate.
72
What effect does infinite [S] have on Competitive inhibition?
Results in the inhibitor having no effect.
73
Why is high [S] not effective at stopping Mixed or noncompetitive inhibition?
The inhibitor does not directly compete with S therefore it can still bind to the enzyme at a different binding site than the S binding site.
74
Uncompetitive Inhibition Mechanism?
Inhibitor only binds to the ES complex.
75
What is Methotrexate?
Similar to folic acid.
Strong competitive inhibitor of dihydrofolate reductase, shuts down DNA synthesis.
76
What is Arsenite?
Noncompetitive Inhibitor
Blocks the catalytic activity of lipoamide containing enzymes such as the PDH and a-ketoglutarate dehydrogenase.
77
What are Statins and what do they do?
Inhibitors of Cholesterol Biosynthesis.
Aka, HMG - CoA reductase inhibitors, are a class of drugs used to lower cholesterol levels, as they are structural analogs of the natural substrates.
78
What is Penicillin?
It is a Irreversible inhibitor of Transpeptidase active-site Ser.
It results in a covalent acyl - enzyme product. This is hydrolyzed so slowly as to be practically irreversible.
79
What is Fluorouracil?
It is a irriversible inhibitor of DNA synthesis, used to treat skin cancer.
80
To determine enzymatic activities what conditions are assays performed under?
To determine what?
At saturating substrate concentrations [S]
To determine the maximum enyme activity ( The point at which the velocity of an enzymatic reaction (v) is only proportional to enzyme activity and/or conenctration [E]. **Zero point kinetics**).
81
How are assays performed to determine kinetic parameters such as Km and rate constants?
The assays are performed **at limiting [S] concentrations.**
This corresponds with the linear region of M - M plot (**First Order Kinetics**).
82
NAD(P) - dependent dehydrogenase reactions are ideal for what?
and how does it work?
Common example:
Rapid analysis of multiple samples.
Reduction of NAD+ or NADP+ produces a new, broad absorption band with a maximum at 340 nm.
LDH
83
In what ways may isozymes differ?
Specific Regulation
Reaction Rate
Electrophoretic Mobility
Immunologic Properties
84
Release of isozymes from different tissues into serum may indicate what?
How is it detected?
Organ damage
Colorimetric assay
85
Electrophoretic separation of proteins quantifies what?
The protein amount not the activity.
86
Regulatory Enzymes definition
Have special properties leading to regulatory roles in metabolism.
87
Regulation of enzymes activities can be achieved by?
* Noncovalent modification (allosteric)
* Covalent modification
* Reversible
* Irreversible
88
Feedback Inhibition
First enzyme in a multi-step pathway inhibited by the final product of the pathway.
89
What type of enzymes are usually noncovalently regulated?
Oligomeric enzymes
90
How does Allosteric enzyme regulation work?
Modulator binds non-covalently to site other than active site
negative modulators inhibit; positive stimulate.
91
Homotropic allosteric regulator?
Is a substrate for it's target enzyme.
Eg. O2 for hemoglobin
92
Hetertropic Allosteric regulator?
A regulatory molecule that is not also the enzyme's substrate.
It could be an activator or an inhibitor of the enzyme.
Eg. CTP and ATP for aspartate transcarbomylase.
93
What is the shape of Allosteric enzymes V0 vs [S]?
The graph is Sigmoidal.
94
What type of Allosteric regulator or modulator is ATP?
Heterotropic positive
95
What type of Allosteric regulator or modulator is CTP?
Hetertropic Negative
96
What are some examples of covalent modifications or post translational modifications (PTMs) of metabolic enzymes?
Phosphorylation, and acetylation of enzymes
97
Examples of regulatory post translational modifications of amino acid side chains?
Phosphorylation, Acetylation, methylation, and ADP - Ribosylation
Often Reversible
98
Some examples of post-translational structural modifications are?
Prenylation, Glycosylation, hydroxylation, and fatty acid acylation
99
Proteolysis
Irreversible regulatory destruction of protein.
100
What type of enzyme adds a phosphate group to the hydroxy side chains of Ser, Thr, and Tyr.
Kinases
101
What type of enzyme removes phosphorylation?
Phosphatases
102
Kinases add a phosphate group to the hydroxyl side chains of what amino acids?
Ser, Thr, and Tyr.
103
Up to ____ of proteins are regulated by reversible phosphorylation.
70%
104
How does phosphorylation regulate proteins?
Two negative charges on these polar side chains regulate the structure and function of proteins.
105
