Lecture 9-10: Theisen Flashcards
(109 cards)
1
Q
Oxioreductases
A
Transfer electrons from a donor to an acceptor
2
Q
Transferases
A
Transfer a functional group between molecules
3
Q
Isomerases
A
Rearrange/isomerize molecule
Intramolecular
4
Q
Lyases
A
“Synthases” add or remove atoms to or from a double bond (from water, ammonia, CO2)
5
Q
Ligases
A
Synthetases-form bonds w/ hydrolysis of ATP
6
Q
Hydrolases
A
Cleave bonds via the addition of water-hydrolysis
Transfers functional groups to water
7
Q
Catalysts
A
Increasing the rate of chemical reactions
8
Q
How do enzymes affect energy of activation?
A
By forming an enzyme-substrate complex—lowering the energy of activation
9
Q
Enzymes get substrates to their:
A
Transition state
10
Q
Uncatalyzed rate w/ carbonic anhydrase
A
Significantly slower [ 10-1 vs 10^6] -1,000,000 X / sec
11
Q
Sir Archibald Edward Garrod
A
1st making connection btw disease and fundamental errors in biochem. Rxn’s. [alkaptonuria] coined the term inborn errors of metabolism
12
Q
2 Reaction drivers:
A
Mass action [Le Chatelier’s Principle]
Coupled reactions [input of energy]
13
Q
MAss action / Le Chatelier’s
A
Increase in [ ] of products= more reactants
Increase [ ] of reactants = more products
Opposite direction from adjustment made
14
Q
Coupled reactions [input of energy]
A
Coupling rxn’s is possible if they share a common intermediate
15
Q
What increases rxn velocity?
A
Enzymes
16
Q
What indicates whether the rxn will proceed w/ or w/o input of energy? Spontaneous
A
Gibbs free energy deltaG
17
Q
What Gibbs energy is favored?
A
Negative
18
Q
Oxidation-reduction reactions
A
Electron donor will reduce the electron acceptor (reducing and oxidizing agents)
19
Q
Acid-base rxn’s
A
Weak acid dissociates in water==releases proton and conjugate base
Weak base combines w/ protons in water. Forming its conjugate acid
20
Q
Characteristic of enzymes r/t specificity
A
3D structure and active site.
Specificity of binding depends on the precisely defined arrangement of atoms in the active site.
21
Q
Where does substrate bind to enzyme?
A
Active site
22
Q
Enzyme substrate complex formation: 2 types
A
Lock n key and induced fit
23
Q
Example of enzyme specificity:
A
proteases cleave peptides btw carboxyl and amino group.
24
Q
What limits the reaction rate?
A
Substrate occupying all the available catalytic sites of enzymes
Increasing substrate [ ], reaction rates hit a Vmax.
25
What evidence shows ES complex formed??
X-Ray crystallography. Cytochrome P450 is bound to its substrate camphor- surrounded by residues of the active site and a heme cofactor
26
What is the enzyme active site
3D cleft or crevice formed from the residues of various protein regions an occupies call total volume
Contains a unique microenvironment, usually void of water, and controls the proper shape, pH and polarity for substrate binding and chemical reactivity
27
First law thermodynamics
Conservation of energy. Can't be created or destroyed
28
Transition state
Defined as intermediate structure that is not the substrate and not yet the product----
Unstable and highest free energy
29
Gibbs free energy of activation
The difference in free energy of the transition state and the substrate ----this can be lowered by enzymes
30
Chymotrypsin
Consists of 3 chains. Side chains of the catalytic triad. There are also 2 interstrand and 2 intra strand disulfide linkage.
31
Active AA in Chymotrypsin
Serine- if it is mutated the enzyme will not function.
32
What is the catalytic triad function in Chymotrypsin
Converts serine 195 into a potent nucelophile.
Peptide hydrolysis
33
Mechanism of peptide hydrolysis
Covalent and Acid-base catalysis.
His-57 positions side chain of Ser-195 and to polarize its hydroxyl group so that it is poised for deprotonation. His-57 accepts the proton from Ser-195 hydroxyl group [this is an example of acting as a general base catalyst]
34
His 57 acts as what type of catalyst in Chymotrypsin
General base catalyst
This is because it accepts the H from the OH grp from serine 195
35
Chymotrypsin active site hydrophobic or hydrophilic?
Hydrophobic
36
Cofactor
Small molecule that contributes to the chemical reaction of the enzyme
37
Apoenzyme
Enzyme w/o its cofactor....inactive...Similar to a guitar capo...inactive strings
38
Haloenzyme
Cofactor bound and catalytically active.
39
Are metal cofactors positively or negatively charged?
Positive
40
How do metal cofactors function:
Stable coordination of active site groups-contribute to chemical reactivity
41
Example of metal cofactor:
Zinc activates H2O to form OH nucelophile. 2Histidine-Zn+2-OH2-> 2His-Zn+2-OH-
42
Coenzymes
are small organic molecules- often derived from vitamins
43
Prosthetic group
Cofactor tightly bound to enzyme
44
Cofactor vs. coenzyme
Cofactors are metals; coenzymes are organic [vitamin] based. Coenzymes are cofactors
45
Need to know ex of coenzyme
Tetrahydrofolate (THF)-folate
Source of one-carbon groups for enzymes that transfer them btw molecules.
Vitamin-derived and assists thymidylate synthetase
46
Thiamine pyrophosphate is derived from
Thiamine (vitB-1) transfers 2 carbon groups
47
Pyridoxal phosphate derived from
Pyridoxine (VIT B6)
Transfers amino and carbonyl groups
48
NAD+ and NADP+
Derived from Niacin VIT B-3.
Transfers electrons
49
Ex of disease d.t coenzymes:
Scurvy [lack of VIT C which is cofactor for Lysyl hydroxylase- for collagen assembly-gum and skin disease]
Ariboflavinosis [riboflavin B2-reduced glutathione Reductase activity -Req's FAD] lesion in mouth corners/lips
50
NAD+ is an ex of
Oxioreductase . (Binding site of a Dehydrogenase)
It is cofactor that binds active site in well-defined arrangement for substrate activation
51
Alcohol dehydragenase components function:
NAD is coenzyme
Zn is cofactor -stabilizes coordination of charge residues in a specific arrangement for catalysis......
Acetaldehyde is substrate or product.
NADH is applied electron transfer
52
Overall function of alcohol dehydrogenase
NAD+ is reduced to NADH in the conversion of ethanol to acetaldehyde
53
Fumarase is in what class of enzymes
Hydrates the double bond of Fumarate to malate to make a single bond
Since it removes a bond from fumarate it is a Lyase
54
chymotrpysin is in which enzyme class?
It is involved in peptide hydrolysis- therefore it is a hydrolases
55
Reductant
H or electron donor
56
Oxidant
H or electron acceptor
57
Reducing agent
Gets oxidized
58
Oxidizing agent
Gets reduced
59
Function of alcohol dehydrogenase
Convert alcohol to aldehydes or ketones w/ the reduction of NAD+ (cofactor) to NADH.....
NADH oxidized the ethanol to aldehyde/ketone...oxioreductase
60
Ex of transferase
Kinase: receives functional group from donor-> gives functional group to substrate...
Functional group is phosphate
donor group could be ATP
61
Kinase receptors
Phosphorylate proteins and themselves- transferases----important in signaling
62
Which enzyme class transfers functional groups to water?
Hydrolases-----hydrolysis
63
What is ex of hydrolase that cleaves disaccharide to monosaccharide?
Lactase---via hydrolysis
64
Can add or remove groups to form double bonds WITHOUT WATER
Lyases
65
Ex of Lyase in glycolysis
Aldolase: F-1,6-BP-> DHAP + GAP
66
Enzyme class that creates products which are isomers of the substrate
Isomerases
67
Ex of isomerases:
Triose phosphate isomerases
Intra molecular oxidation-reduction: DHAP-> G-3P
68
Enzyme that uses ATP to covalently bind DNA
DNA ligase- forms covalent phosphodiester bonds btw nucleotides
69
Enzyme kinetics
Study of biochemical reactions that're catalyzed by enzymes...focusing on reaction rates
70
How to determine michalis mention and line weaver burk graph?
From exp. data only
71
Rate of reaction formula
v = (Vmax[S]) / (Km +S])
72
Assumptions of steady-state kinetics
1- ES is in rapid equilibrium
2-Rate of ES formation = rate of ES breakdown
3-[S]>>>[E], S is essentially constant
4- initial velocity is measured at time 0
73
A short description of steady state kinetics:
Affinities
74
Km
Substrate [ ] producing 1/2 Vmax for catalyzed reaction
75
Vmax
Maximum velocity at which enzyme is saturated w/ substrate
76
Michaelis-Menten formula
Vo= Vmax X ([S]/ [S] + Km)
77
Line weaver-burk:
Reciprocal of michaelis-menten
78
Y intercept of line weaver burk
1/ Vmax
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Slope of line weaver burk
Km / Vmax
80
X-int of line weaver burk
-1 / Km
81
Arginine-tRNA synthetase composed of 3 substrate w/ 3 different Km. Why?
Arginine-3
tRNA-0.4
ATP-300
ATP is limiting b/c it is most inefficient substrate. tRNA is lowest Km=most efficient therefore, largest Km=limiting substrate because it must reach 300 for all 3 to function
82
Bisubstrate reactions
Start w/ 2 substrates and end w. 2 products. Most biochemical rxn's and mostly transfer functional group from one substrate to another
83
2 types of bisubstrate reaction
Sequential rxn's
Double displacemnent (ping pong)
84
Sequential reactions:
All substrates bind to the enzyme before any product is released...ex: NADH + pyruvate= lactate + NAD+
85
Double-displacement (Ping pong) reactions
One of the products is released before a second rxn occurs; one product must come off before the rxn can move to next rxn. Ex:Aspartate to OAA/ K-KG, Glutamate
86
Can you apply michaelis menten to allosteric enzymes?
NO!...this has a sigmoidal curve. MM kinetics has hyperbolic curve
87
3 types of enzyme inhibition
Competitive, noncompetitive and uncompetitive
88
Example of an enzyme inhibitor commonly prescribed by MD
Methotrexate---looks much like dihydrofolate but binds 100 fold more tightly
Aspirin---blocks 1st step of prostaglandin synthesis (COX-2 inhibitor)
89
Reversible inhibiton
Binding to enzymes w/ non-covalent interactions: H bonds, hydrophobic interactions, and ionic bonds...do not undergo chem rxn's and can be easily removed by dilution/dialysis
90
Irreversible inhibition
Covalently modifies an enzyme; irreversible. Specifically alter the active site of the protein. EX: FFR-CK covalently binds to active site serine in plasmin.
91
What does the kinetics reveal of an irreversible inhibited enzyme?
Vmax is lowered- d/t less functional enzymes
Km is unchanged- less enzymes doesn't change the functioning ones Km
92
Enzyme inactivation
If continues will eventually lead to 0 Vmax...thus, Vmax decreases, but Km doesn't change...the enzymes are just rendered ineffective by the inactivation-irreversible inhibition
93
3 types of reversible inhibition?
Competitive, uncompetitive, and noncompetitive
94
Competitive inhibition
Inhibitors that compete w/ substrates for the active site of the enzyme. Increases Km
95
Uncompetitive inhibition
Inhibition that does not affect the formation of the ES complex but inhibits the enzyme at site other than the active site (allosteric binding site). Since binds to EScomplex... Km and Vmax affected
96
Noncompetitive inhibiton
Inhibitors that can bind w/ both the enzyme and ES complex. Since it is not inhibiting enzyme function, it only affect Vmax(decreases).
97
V max and Km for Competitive inhibition:
Vmax unchanged; Km increased
Vmax doesn't change b/c enzymes still functioning.
Km increases b/c decreased Km would be MORE efficient. When there are 2 competing for the same enzyme...the efficiency will suffer.
98
V max and Km for Uncompetitive inhibition
Vmax decreases; Km decreases
Vmax decreases b/c enzymes are beginning to be unable to function.
Km decreases b/c uncompetitive binds to ES complexes...thus Enzyme and substrate have been inactivated.
99
V max and Km for NONcompetitive inhibition
Vmax decreased; Km unchanged
Vmax is decreased b/c the inhibitor lowers the [] of functional enzyme
Km is unchanged because there is still enzyme that is functional in sol'n
100
pH and activity
Sigmoidal curve w/ low Kcat @ 6 and higher point @ 9 pH...pH affects shape and function of proteins(denaturation @ low pH)---this changes function b/c shape changes effect substrate binding and activity
101
Pepsin vs. trypsin in pH
Pepsin optimally functions at low pH(2) in stomach
Trypsin is further down GI tract and functions optimally at higher pH [8]
Resembles a bell curve....this can be done for temperature too.
102
The jist of inhibiton for these lectures: 2 things:
```
Covalent modification (irreversible-signaling)
vs.
allosteric inhibiton (Enzyme-substrate-reversible)
```
103
Common covalent modification:
phosphorylation
104
What part of the enzyme dictates specificity
Active site
105
Enzyme inactivation: irreversible inhibition
Vmax decreased; Km unchanged
Vmax decreased=less functionality enzyme
Km = the functional enzyme that is still available Km doesn't change
106
Can bind w/ Enzyme and ES complex:
Noncompetitive inhibition
107
Inhibition that doesn't affect the formation of the ES complex but inhibits the enzyme at a site other than their active site. Only binds to ES complex.
Uncompetitive inhibiton
108
Inhibitors that compete w/ substances for the active site of the enzyme.
Competitive inhibition
109
What is ATCase 's substrate?
Aspartate
