week 3 Flashcards
(141 cards)
1
Q
which biomolecule is the basis for nucleic acid?
A
nucleotides.
2
Q
what are the types of nucleic acids?
A
1) DNA: deoxyribonucleic acid
* forms the inherited genetic material inside our cells.
§ Segments of this DNA, called genes, code for
protein & determine our physical traits
§ 2) RNA: ribonucleic acid
3
Q
What is the central dogma?
A
- The central dogma of molecular biology is a theory stating that genetic information flows only in one direction, from DNA, to RNA, to protein, or RNA directly to protein.
- DNA does not direct protein synthesis itself but uses RNA as an intermediate.
4
Q
What are the 3 components of a nucleotide?
A
- phosphate group.
- deoxyribose sugar and a nitrogenous base.
5
Q
what are the 2 types of nitrogenous bases?
A
- pyrimidines; single ring base.
- purines; double-ring base.
6
Q
which of the 4 bases are purines?
A
adenine and guanine.
7
Q
which of the 4 bases are pyrimidines?
A
cytosine and guanine.
8
Q
outline the DNA structure;
A
- The 3D structure of DNA is
a double helix:
§ The sugar-phosphate
backbone forms a righthanded double helix to
maximize efficiency of the
base-pairing - FYI - 1 complete turn for
every 10 base pairs - The two DNA strands are
held together by H-bonds
between bases - A pairs with T
- G pairs with C
9
Q
why do A and T always combine and why do C and G always combine?
A
- A always pairs with T and G always pairs with C because these are the only combinations that allow for hydrogen bonding to occur, given the spatial constraints of the double helix, which requires there to be one purine and one pyrimidine in each base pair.
10
Q
why does the number of purines have to equal the number of pyrimidines?
A
-According to Chargaff’s rule, the number of purines and pyrimidines exist in ratio 1: 1. This is so because purines bond with pyrimidines to form nucleotides hence in a double-stranded DNA, the number of purines is equal to the number of pyrimidines.
11
Q
what forces are needed to stabilize the DNA double helix?
A
- H-bonds between complementary base pairs
* 2. Sugar phosphate backbone
§ More on next slide
* 3. Base stacking
12
Q
what is the importance of the sugar phosphate backbone?
A
- the sugar phosphate back bone is connected by phosphodiester bonds.
13
Q
What are nucleosomes?
A
- Nucleosomes are the structural unit for packaging DNA.
14
Q
what are nucleosomes composed of?
A
- 147 base pairs wrapped around a histone core.
- octamer of H2A, H2B, H3 and H4.
- H1 linker protein.
15
Q
what is chromatin?
A
- complex of DNA and tightly bound protein.
- it can be found as either densely packed heterochromatin or dispersed euchromatin.
16
Q
which type of chromatin is found when the cell is transcriptionally active?
A
- Heterochromatin is highly condensed, gene-poor, and transcriptionally silent, whereas euchromatin is less condensed, gene-rich, and more easily transcribed
17
Q
What are chromosomes?
A
- in its most condensed form DNA is packaged into chromosomes.
- most humans cells have 23 pairs of chromosomes (46) total.
- one copy of each chromosome comes from each parent. (2n; diploid).
- the maternal and paternal chromosome pair are called homologous chromosomes (aka homologs).
18
Q
what cells in the human body are haploid meaning they contain only 1 copy of each chromosome?
A
- sex chromosomes.
19
Q
what are genes?
A
- chromosomes carry genes the functional units of heredity.
- a segment of DNA containing the instructions for making a particular protein.
20
Q
what is the difference between a exon and a intron?
A
- exon= coding sequence of a gene.
- intron= non-coding sequence of a gene. they are removed via splicing after transcription.
21
Q
What is non-coding DNA?
A
- 98.5% of the human genome does not encode protein.
- a large majority of this non-coding DNA regulates gene expression.
22
Q
how does non-coding DNA regulate gene expression?
A
- promoter and enhancer regions; bind transcription factors.
- binding sites for factors that organize chromatin structures.
- non-coding regulatory RNA eg. microRNA.
- mobile genetic elements (transposons) not well understood. implicated in gene regulation and chromatin organization.
23
Q
What is the template strand?
A
- Like DNA, RNA is a polymer of nucleotides linked by a phosphodiester bond.
24
Q
how does DNA differ from RNA chemically?
A
- ribose sugar vs deoxyribose sugar.
- uracil base rather than thymine.
- still contains adenine, guanine and cytosine.
25
how does DNA differ from RNA structurally?
- DNA always occurs in a double stranded helix.
- RNA is single-stranded.
- Therefore it can fold up into various shapes.
26
What is mRNA?
- DNA is transcribed into RNA to serve as a template for protein translation.
- in this case the RNA transcript is called messenger RNA (mRNA).
- initially it is termed pre-mRNA.
- Pre-mRNA then undergoes processing into mature mRNA.
-
27
What is non-coding RNA?
- RNA that does not get translated into proteins.
-non-coding RNA serve as enzymatic, structural, and regulatory components of a wide variety of processes in the cell.
28
What is snRNA?
- type of non-coding RNA.
- Small nuclear RNA functions in the spliceosome.
- spliceosomes are needed to remove introns from pre-mRNA.
- snRNA is associated with protein subunits to form small nuclear ribonucleoproteins (snRNPs) which form the core of the spliceosome.
29
What is rRNA?
- ribosomal rRNA
- this is needed for the basic structure of the ribosome complex.
- it is involved in the catalysis of the peptide bond between amino acids.
30
What is tRNA?
- transfer RNA are needed in translation to carry the correct amino acid to the growing polypeptide chain.
- tRNA folds into a unique cloverleaf shape.
31
What parts of the tRNA are particularly important to protein translation?
1. the anticodon; 3 consecutive nucleotides that pairs with complementary codon in an mRNA molecule.
2. amino acid binding site; short single-stranded region at the 3' end of the tRNA molecule.
- binds the amino acid that corresponds to the anti-codon on the tRNA.
32
What is the wobble hypothesis?
- despite 64 possible combinations of nucleotides into a 3 nucleotide codon.
- there are only 20 amino acids.
- this demonstrates the redundancy of the genetic code.
- this implies that there is more than one possible tRNA for many of the amino acids OR.
- some tRNA molecules can base-pair with more than one codon--> both are true.
- some tRNA are built to only require accurate base-pairing of the first two-positions of the codon and can tolerate mismatch (AKA wobble) in the third position.
- this explains why so many of the alternative codons for an amino acid only differ in their 3rd nucleotide.
33
What is miRNA?
- non-coding RNA
- micro RNA.
- regulates gen expression via psot-transcriptional silencing.
- blocks/prevents translation of specific MRNAs and promotes their degradation.
34
What is siRNA?
- small interfering RNA
- non-coding RNA.
- reduces gene expression; direct degradation of specific mRNA
35
what is incRNA?
- long non-coding RNA.
- regulates gene expression.
- can increase or decrease transcription.
- involved in X-chromosome inactivation.
36
what is transcription?
- transcription refers to the process of synthesizing an RNA molecule from DNA template (gene) that will dictate the synthesis of a protein.
- occurs in the nucleus.
37
what is the transcriptional unit?
- the transcription unit outlines the 3 general regions found in all geins.
1. promoter region; contains consensus sequence.
2. coding region; transcribed into mRNA.
3. terminator region; specifies end of transcription
38
What is the template strand?
- the DNA strand that is transcribed into RNA is referred to as the template strand. it can also be referred to as the anti-sense strand.
- the template strands complimentary partner is referred to as the non-template strand.
- it can also be referred to as the sense strand.
39
what is RNA polymerase?
- it is the main key enzyme for transcription.
- it moves along the DNA unwinding the DNA helix just ahead of the active site for polymerization.
- it catalyzes a new phosphodiester bond on the newly-forming strand of RNA.
- RNA polymerase works in the 5' to 3' direction (complementary stand).
40
outline initiation of transcription;
- in order to begin transcription, RNA polymerase must recognize where to start.
- transcription initiation factors help with this process.
- in prokaryotes there is just one (sigma).
- in eukaryotes there are many different types, we will consider the role of general transcription factor TF11.
- needed for RNA polymerase 11 which transcribes all protein-coding genes in eukaryotes.
41
initiation A;
- TF11 recognizes and binds a consensus sequence in the promoter region.
- in eukaryotes one example is called the TATA box. located 25 nucleotides upstream from the transcription start site.
- TF11D is the specific TF11 that binds the TATA box.
42
initiation B,C,D
B). other transcription factors join.
C). RNA polymerase 11 joins.
D). transcription initiation complex is complete and transcription can begin.
43
outline transcription regulation;
- The TATA box ( or other consensus sequences) aren't the only binding sites on DNA that influences initiation of transcription.
- repressor proteins bind upstream sequences called silencers (aka negative regulatory elements). they inhibit gene transcription.
44
what are transcriptional activator proteins)
- they bind upstream sequences of DNA called enhancers (aka positive regulatory elements).
- increase the rate of transcription by attracting the RNA polymerase 11 enzyme.
45
outline elongation;
- Once RNA polymerase begins transcribing DNA most of the general transcription factors are released.
- these transcription factors are then available to initiate another round of transcription with new RNA polymerase molecule.
- RNA polymerase moves downstream along the DNA, transcribing the coding region.
- various elongation factors are needed to help reduce the likelihood that RNA polymerase dissociates from DNA before it reaches the end of the gene.
46
in addition to elongation factors what do eukaryotes also require?
- chromatin remodelling complexes and histone chaperones.
47
what are chromatin remodelling complexes?
- hellp the RNA polymerase navigate the chromatin structure.
48
what are histone chaperones?
- partially disassemble and reassemble nucleosomes as an RNA polymerase passes through.
49
true or false? as RNA polymerase moves along the DNA double helix it generates supercoils.
- yes in eukaryotes DNA topoisomerase removes this super-helical tension.
50
what is DNA topoisomerase?
- The enzyme DNA topoisomerase relieves the super-helical tension by breaking the phosphodiester bond.
- this allows the two sections of the DNA helix to move freely and relieve tension.
- The phosphodiester bond will re-form as DNA topoisomerase leaves.
51
outline processing;
- in eukaryotes during elongation the pre-mRNA transcript is processed in 3 main ways.
1). Splicing.
2). Capping the 5' end.
3). polyadenylation of the 3' end.
- once these modifications are complete the transcript is called mRNA.
52
what is capping of the 5' end?
- part of processing.
- a modified guanine nucleotide is added to the 5' end of the transcribed pre-mRNA.
- this occurs early once 25 nucleotides of RNA have been transcribed.
- This 5' cap facilitates export of the mRNA into the nucleus and is involved in translation.
- 7-methyl guanosine cap.
53
What is splicing?
- Both intron and exon sequences are transcribed into RNA.
- introns are then removed in a process called RNA splicing.
- introns are then removed in a process called RNA splicing.
- spliceosomes require a special form of RNA (snRNA) and proteins complexed into snRNPs.
- SnRNA= small nuclear RNA.
- snRNP= small nuclear ribonucleoprotein.
- snRNP is referred to a spliceosome once it has complexed with the pre-mRNA.
54
Why does splicing occur?
- 95% of human genes are spliced in more than one way.
- splicing allows the same gene to produce a variety of different proteins.
55
outline processing and termination;
- the 3' end of the mRNA molecule is specified by signals encoded in DNA.
- these signals are transcribed into RNA and then bind to proteins that facilitate cleavage of mRNA from RNA polymerase.
56
what is polyadenylation of the 3' end?
- once cleaved 200 A nucleotides are added to the mRNA.
- this is catalyzed by a enzyme called poly-A polymerase (PAP).
- poly A tails protect the mRNA from degradation and facilitates export from the nucleus.
- Poly A binding proteins then bind the poly-A tail.
57
what is transcription like in prokaryotes?
- the steps of transcription in prokaryotes are the same, however, the mRNA transcript produced in prokaryotes is a little different.
- no processing is required for the prokaryotic mRNA transcript.
- no 5' cap, splicing, or poly-A tail.
- no export from the nucleus, thus translation can begin right away.
- mRNA transcript is polycistronic. codes for more than one protein.
58
What is translation?
- following processing and termination of transcription, mature mRNA is exported from the nucleus through nuclear pore complexes.
- once in the cytosol mature mRNA is translated into protein.
59
What happens to the mRNA during translation?
- The mRNA sequence is decoded in sets of 3 nucleotides called codons.
- there are 64 possible combinations of 3 nucleotides but only 20 amino acids..
- some amino acids are specified by more than one codon, demonstrating the redundancy of the genetic code.
60
what are reading frames?
- since mRNA is interpreted in 3- nucleotide codons, the correct polypeptide sequence depend on the correct reading frame.
- special punctuation is needed to determine the correct reading frame.
- in eukaryotes the first sequences is AUG (methionine).
- in prokaryotes it is the shine dalgarno sequence.
61
What molecules are needed for translation?
- mRNA transcript.
- tRNA. needs to be bound to the correct amino acids (charged tRNA).
- ribosomes ( a large and small subunit).
62
outline preparing the tRNA
- the cell makes a variety of tRNAs each corresponding to one of the 20 amino acids.
- the enzyme aminoacyl-tRNA synthetase catalyzes the attachment of the correct amino acid to tRNA.
63
What is needed for translation?
- protein synthesis is performed in the ribosome.
- helps to maintain the correct reading frame and ensures accuracy of the codon- anti-codon interaction.
- The ribosomal complex is composed of various ribosomal proteins and ribosomal RNA (rRNA).
- there is a large and small ribosome subunit.
64
what are the three steps of translation?
1. initiation.
2. elongation; tRNA binding, peptide bond formation, large subunit translocation, and small subunit translocation.
3. termination.
65
outline translation initiation;
- AUG is the first codon translated on the mRNA.
- initiator, tRNA carries the amino acid methionine.
- this forms a initiator tRNA-methionine complex (Met-tRNAi)
- Met-tRNAi is loaded into the small ribosomal subunit with initiation factors.
- small ribosome binds to the 5'end of the mRNA. the 5'7-guanosine cap helps with recognition of the 5' end.
- small ribosome moves along the mRNA (from 5' to 3') scanning for the first AUG. requires ATP hydrolysis.
- initiation factors dissociate and the large ribosomal subunit assembles to complete the ribosome complex.
66
outline prokaryotic initiation of translation.
-prokaryotic mRNA is polycistronic.
- an additional recognition sequence is needed for ribosome binding. the shin-dalgarno sequence (aka ribosome-binding site).
67
outline elongation of translation;
A). tRNA binding; newly charged tRNA binds to the A site of the ribosome complex.
B). peptide bond formation; carboxyl end of the polypeptide chain is released from the tRNA at the P site and joins the amino acid linked to the tRNA at the A site.
- this new peptide bond is catalyzed by peptidyl transferase enzyme contained with in the large ribosomal subunit.
C). translocation of the large subunit; large relative subunit moves relative to the mRNA held by the small subunit.
- Two tRNAs are shifted to the E and P sites.
D). translocation of the small subunit.
- small subunit shifts by 3 nucleotides.
- tRNA in E site is rejected.
- cycle is then repeated for a new incoming acyl-tRNA
68
how do elongation factors help with translation?
- elongation proceeds efficiently and accurately with the help of elongation factors.
- these elongation factors enter and leave the ribosome during each cycle and are coupled with GTP hydrolysis.
69
outline termination of translation;
- A stop codon marks the end of translation.
- UAA, UAG, UGA
- not recognized by tRNA and do not specify an amino acid.
- release factors bind to ribosome with a stop codon in the A site.
70
What are polysomes?
- synthesis of proteins occurs on polyribosomes or polysomes.
- multiple initiations take place on each mRNA molecule being translation.
As soon as the preceding ribosome has translated enough of the nucleotide sequence to move out of the way, a new ribosome complex is formed.
Helps speed up rate of protein synthesis
71
what happens post translation?
- protein is folded into specific 3D shape.
- proper folding is important because structure of protein dictates its function.
- more to come next class.
- may be modified in the ER; glycosylated; addition of mono or oligosaccharide--> sent to its proper cellular location.
72
How is transcription regulated?
- histones.
73
what are histones?
- histones are highly dynamic structures regulated by a host of nuclear proteins.
74
what are chromatin remodelling complexes?
- can reposition nucleosomes on DNA to either expose or obscure gene regulatory elements. (promoters).
75
what are chromatin writer complexes?
- can carry out histone modification such as methylation, acetylation or phosphorylation.
76
What is histone acetylation?
- tends to open chromatin and increase transcription.
- performed by histone acetyltransferases (AKA HAT).
- deacylation reverses these changes and promotes chromatin condensation.
77
what is histone methylation?
- histones and DNA can both be methylated.
- histone methylation can promote transcriptional activation or repression. (depends on the histone residue).
- DNA methylation typically results in transcriptional silencing.
78
What is mRNA stability?
- the longer a mRNA lasts in the cytosol the more protein will be made via translation.
- remember that some of the non-coding forms of RNA (miRNA or siRNA) could promote the destruction of an mRNA transcript.
- specific proteins can also bind to mRNA and prevent its degradation to results in more protein synthesis.
- example; transferrin; protein receptor that brings iron into a cell.
79
What is targeting to cellular location?
- a cell can limit a protein to a particular cellular location.
- proteins needed for intracellular cytosolic use are translated on free ribosomes in the cytosol.
80
what happens to proteins destined for the nucleus, mitochondria or peroxisomes?
- once translated into the cytosol, a specific amino acid signal in the polypeptide will target the protein for its intracellular location
81
What happens to proteins destined for lysosomes, cell membrane or secretion
- They need to be directed to the rough ER for translation in a process called co-translational transfer.
- translation begins off a free ribosome in the cytosol.
- a signal peptide sequence is translated, which binds a signal recognition particle (SRP).
- binding of SRP stops translation and directs the ribosome to the RER when it binds to a SRP receptor.
- translation re-starts, with the growing polypeptide moving through a channel into the lumen of the RER.
- once in the RER, the signal peptide sequence is removed.
82
What happens if the protein needs to be inserted into a cell membrane?
- it will contain a stop transfer sequence.
- when the stop transfer sequence comes into contact with the translocator, translation is paused.
- translocator will discharge the polypeptide into the phospholipid bilayer of the ER membrane.
83
what are the two shapes of proteins?
- fibrous and globular.
84
outline fibrous proteins;
- long- and rod-shaped.
- generally has a structural function/ provides strength.
- often insoluble in water.
- keratin and collagen.
85
outline globular proteins;
- compact and spherical.
- generally has dynamic functions.
- eg. enzymes to catalyze reactions.
-eg. carrier proteins.
- often soluble in water.
- enzymes, albumin, hemoglobin.
86
what are the different compositions of proteins?
- simple or conjugated.
87
what are simple proteins?
- composed of only amino acids.
88
what are conjugated proteins?
- composed of protein and non-protein portion.
- protein portion; contains only amino acids.
- non-protein portion; called prosthetic group, a conjugated protein without its prosthetic group is called a apoprotein.
89
What are the 4 protein structures?
1. primary.
2. secondary.
3. tertiary.
4. quaternary.
90
What is primary protein structure?
- Primary protein structure
Polypeptide chain
Linear sequence of amino acids
Synthesized via translation from mRNA transcript derived from a gene (DNA)
Amino acids are held together via peptide bond
91
what is secondary protein structure?
Regularly repeating backbone conformations formed by H-bonds between carboxyl and amino groups
Two main types
Alpha helix
Beta pleated sheet
92
What is a alpha-helix?
-Alpha Helix:
Each carboxyl group H-bonds with an amino group 4 amino acids away
Forms rigid, rod-like structures
Often depicted schematically like piece of curled ribbon
93
what is a beta-pleated sheet?
beta-pleated sheet
Two or more polypeptide segments of a protein line up side-by side
Held together by H-bonds between distant carboxyl and amino groups
Often depicted schematically with tip pointing in C-terminal direction
94
what is a super-secondary structure?
Super-secondary structure within a protein are combination of alpha helices and/or beta-pleated sheets
Here are some examples:
- helix-loop-helix.
- beta-alpha-beta.
- B-meander.
- beta-barrel.
95
what is the tertiary structure?
Three-dimensional folded structure created by side chain interactions, such as:
H-bonds
Salt bridges
Disulfide bridges
Hydrophobic interactions
96
why are disulfide bonds important for tertiary structure?
Disulfide bonds are very important in extracellular proteins
Strong bonds: help protect the protein from denaturation during changes in blood pH or salt concentrations
What is an example of an extracellular protein that is held together by disulfide bonds? insulin
97
what is the quanternary structure?
Many proteins have multiple polypeptide subunits
The association of all the subunits form the quaternary structure of a protein
Now it is a functional protein!
Consider hemoglobin:
4 subunits; 2 beta and 2 alpha.
The final 3-D shape of a protein dictates its function
98
what are the different types of quanternary structures?
A protein composed of two subunits is called a dimer
A protein composed a several subunits is called an oligomer
Hemoglobin is an example of an oligomer
A protein composed of many subunits is called a multimer
A protomer is any repeating structural unit within a multimeric protein
Hemoglobin has a pair of αβ protomers
99
outline protein folding;
Following protein translation, proteins are folding into their secondary, tertiary, and quaternary shapes.
Chaperones are proteins that help other proteins:
Fold into their correct shape
Get to their correct cellular locations
Common chaperones are the hsp (heat shock proteins) which can:
Bind and stabilize portions of the protein not yet folded
Chaperones are eventually released via ATP hydrolysis
Refold proteins partially unfolded due to stress
100
What is protein denaturation?
- lets consider the loss of protein structure, which is disruption in its folding or shape.
- occurs when the bonds holding the protein bonds together are disrupted.
101
how can bonds within proteins be denatured?
Bonds within proteins can be disrupted and the proteins denatured by using:
Strong acids or bases, or reducing agents:
Add or remove hydrogens
Organic solvents, detergents:
Disrupt hydrophobic, polar and charged interactions
Salts:
Disrupt polar and charged interactions
Heavy metal ions
102
How do heavy metals disrupt the protein bonds?
As well as binding to negatively charged groups, heavy metals can also bind to sulfhydryl (SH) groups
This alters the shape of the protein
This is the basis of “lead poisoning”
Pb+2 binds to the SH groups in two enzymes needed for the synthesis of Hb
Lack of Hb synthesis leads to the hallmark anemia associated with lead poisoning
103
what are enzymes?
- Enzymes are an example of a type of globular protein.
Most biochemical reactions that occur in our body require enzymes in order to occur at a pace fast enough to support life.
- Enzymes = protein catalysts that speed up specific reactions and remained unchanged by the reaction
Enzymes speed up a reaction by lowering the activation energy of the reaction without changing:
The standard free energy (ΔG) of the reaction
The equilibrium of the reaction
They speed up a specific reaction by lowering the activation energy Ea
104
what is activation energy?
The minimal amount of energy needed to make/break the bonds necessary for a reaction to occur
Also known biochemically as the free energy of activation (ΔG‡)
Sometimes defined as the amount of energy needed to reach the transition state (TS)
The transition state is the highest energy configuration formed when changing from reactants to products
Transient and not isolated
105
even tho activation energy is lowered does this influence the standard free energy of the reaction?
- No change in overall standard free energy of the equation (ΔG)
106
what is enzyme specificity?
Enzyme molecules contain a special cleft called the active site
The active site forms by precise quaternary structure of the protein
Amino acids in the active site participate in substrate binding and catalysis
Enzymes are highly specific
Only a substrate of the correct size and shape can enter into the active site.
107
What is the ES complex?
Once inside the site, the substrate binds the enzyme forming an enzyme-substrate (ES) complex
108
what is the induced fit model?
Binding of substrate is thought to induce a conformational change in shape of the enzyme
This is called the Induced fit model
109
what are the mechanisms of a enzyme?
Once a substrate binds to the active site, how exactly does it speed up a reaction?
The induced-fit between the correct substrate and enzyme allows for:
Electrostatic interactions to form between the two
The correct positioning of catalytic groups in the enzyme
Catalytic groups may speed up reactions in two main ways
Acid-base catalysis
Covalent catalysis
110
what are the acid-base effects of enzyme mechanics?
Addition or removal of a proton can make a substrate more reactive
The side chains of certain amino acids can add or remove H+’s by acting as general acids or bases
111
outline acid base catalysis from slow to fast.
1. slow= no acid base.
2. fast= acid or base catalysis.
3. very fast= acid + base catalysis.
112
what is covalent catalysis?
A nucleophilic side group in the enzyme active site forms a temporary covalent bond with the substrate
Common nucleophilic side groups include:
Asp and Glu (R-COO-)
Ser (R-OH) and Cys (R-SH)
Serine and Cystein are only weakly nucleophilic, but their nucleophilicity is enhanced by the presence of other amino acids that can remove the H
113
what are cofactors?
Enzymes often have help from cofactors and coenzymes
Cofactors are typically metal cations
Mg2+, Zn2+
Magnesium helped to position the ATP in the enzyme active site
Helped to stabilize the negative charges on the ATP
114
What are coenzymes?
Enzymes often have help from cofactors and coenzymes
Coenzymes
Typically derived from vitamins B3: NAD+ NADH + H+
Can accept or donate elections in redox reactions
115
how do coenzymes and cofactors speed up the rate of reaction?
In summary, cofactors and coenzymes can help enzymes speed up reactions in three main ways:
Can help position the substrate in the active site of the enzyme
Can stabilize negative charges on the substrate or the TS to make it easier for a nucleophilic attack to occur
Can accept/donate electrons in redox reactions
116
how does temperature effect enzymes?
The optimal temperature for an enzymes is usually the temperature of the organism
117
how does pH effect enzymes?
Changing the pH can change the protonation state of the enzyme and/or the substrate
What types of bonds between E and S would potentially be disrupted by a change in pH?
H-bonds – for example:
If an H is removed, no H-bond can be formed
If an H is added, an H-bond might form that is not usually formed.
Electrostatic interactions - for example:
Adding an H can turn COO- into COOH
Removing an H can turn NH3+ into NH2
118
what are 4 ways the enzyme is controlled?
1. Genetic
2. Covalent modification
3. Allosteric regulation
4. Compartmentalization
119
what is genetic regulation of an enzyme?
Enzymes transcription can be induced or repressed based on the needs of the cell
In times of needs enzyme transcription and translation will be induced
In times of excess enzyme transcription and translation will be repressed
120
what is an example of genetic regulation?
- when you consume a high carbohydrate meal insulin will go up.
- this increases the transcription of glucokinase, pFK1 and pyruvate kinase so we end up getting more translation and then more of these enzymes in the cytosol.
- resulting in more efficient conversion of glucose to pyruvate.
121
What is covalent modification?
Involves altering the structure of an enzyme (or “proenzyme”) by making or breaking covalent bonds
There are two types of covalent modification:
Reversible
Irreversible
122
what is reversible covalent modification?
Reversible covalent modification
Involves addition or removal of a group to the enzyme that causes it to convert to its active or inactive form
What is a common group that can do this?
Does addition of this group activate or inactivate the enzyme?
Other common groups that can be added to or removed enzymes include methyl and acetyl groups
123
what is an example of reversible covalent modification?
Example – glycogen metabolism
The main regulated enzyme in glycogenesis is de-activated by phosphorylation while the main enzyme in glycogenolysis is activated by phosphorylation.
Phosphorylation is catalyzed initially by the same protein, protein kinase A (PKA)
This prevents both pathways from running at the same time
124
what is irreversible covalent modification?
- Irreversible covalent modification
Involves cleavage of peptide bonds in proenzymes or zymogens
Makes sure the enzyme is not used until it is in the correct location and until it is needed
125
what is allosteric modification?
-Allosteric modification of allosteric enzymes
Binding to enzyme’s allosteric site changes the conformation and activity of the enzyme
Changes the binding affinity of the substrate at the active site
commonly used to control regulatory enzymes.
Allosteric enzymes have more than one subunit
Allosteric site is on one subunit, active site on another
The binding of an effector molecule to an allosteric enzyme can either:
Increase binding of the substrate to the enzyme
Effector = activator
Decrease binding of the substrate to the enzyme
Effector = inhibitor
126
what is an example of allosteric modification?
Consider the glycolytic enzyme phosphofructokinase-1 (PFK-1):
What reaction is catalyzed by PFK-1? It is reversible or irreversible?
Phosphofructokinase-1 is inhibited allosterically by high levels of ATP
Can you think of why this might be the case?
Phosphofructokinase-1 in activated allosterically by high levels of AMP
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what is compartmentalization?
Compartmentalization of enzymes via membrane-bound organelles allows for regulation by:
1. Separation of enzymes from opposing pathways into different cellular compartments, and selective transportation of substrates.
2. 2. Creation of unique microenvironments
Lysosomal enzymes function at a pH around 4.5-5, while most other cellular enzymes function at a pH around 7.
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what are Michaelis-Menten Kinetics?
Michaelis and Menten (M & M) developed a kinetics equation for certain reactions*
What do we use it for?
To determine if an enzyme is physiologically useful based on its:
Maximum rate
Affinity for substrate (or inhibitor or coenzyme…)
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what are 1st order reaction?
- M & M kinetics applies to first order reactions
1st order: Concentration of a single substrate is directly proportional to the rate of the reaction.
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what is a first order reaction with two substrates called?
-This type of reaction is called a “pseudo” first order reaction, and M & M kinetics still apply
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what is the MM equation?
- Vo = Vmax[S] / ([S]+KM).
-Vo: the initial rate of the reaction
[S] is high and [P] is low, therefore no reverse reaction takes place
Km: indication of how well the enzymes binds a given molecule
Small Km = high affinity; large Km = low affinity
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what is the lineweaver-burk graph?
Lineweaver-Burk = reciprocal of M & M equation
Creates a straight line graph, easier to extrapolate
Y-intercept = 1/Vmax
Extrapolated X-intercept = - 1/Km
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what is a zero order reaction?
- Zero order reactions are those in which the concentration of the reactants does not change over time and the concentration rates remain constant.
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What can we use the lineweaver burk plot for?
- Used to determine important terms in enzyme kinetics, such as Km and Vmax, before the wide availability of powerful computers and non-linear regression software. Gives a quick, visual impression of the different forms of enzyme inhibition.
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What is enzyme efficiency?
- Which is a more efficient enzyme: one with a larger or smaller value for Kcat/Km?
- The higher this value the more specific the enzyme is for that substrate. This is because a high value of kcat and a low value of Km are expected for the best substrates.
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What are the different types of enzyme inhibition?
- Reversible inhibition
Competitive, uncompetitive, noncompetitive
Follow M & M kinetics
Irreversible inhibition
Inhibition of allosteric enzymes (multisubunit)
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what is competitive inhibition?
-Competitive inhibition
Reversible binding of the inhibitor to the active site of the enzyme
-Methotrexate
Used for chemotherapy
Competitively inhibits the enzyme that helps convert folate (B9) into its coenzyme form
FYI: E = dihydrofolate reductase
Folate coenzymes normally feed into purine and pyrimidine production – what are these used for?
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how does competitive inhibition effect the km and vmax?
- no change in vmax but a higher km value, meaning lower affinity.
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what is uncompetitive inhibition.
-Reversible binding of I to ES
- the km and v max are both changed, higher km and vmax with the inhibitor.
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what is non-competitive inhibition?
Reversible binding of I to E or ES
Example: Product inhibition
G-6-P inhibition of glucokinase substrate.
- the affinity for the substrate (Km stays the same) but Vmax goes up.
-Binding to E appears to lower affinity, binding to ES appears to increase affinity: cancel out!
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what is reversible inhibition;
Occurs when an inhibitor forms a covalent bind with the active site of the enzyme
