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Year 2: Semester 2 > Biochemistry II > Flashcards

Biochemistry II Flashcards

1
Q

Why are enzymes studied:

A

To understand how they work (for their exploitation using modification in biotechnology and Drug Discovery)
and to understand the effects of mutation on their structure (inherited diseases, conditions, and cancer)

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2
Q

What is a Reaction Mechanism:

A

A diagram describing the flow of electrons through a reaction, from where they are sourced, which bonds are broken, and which are formed and where they end up.

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3
Q

What are the rules of curly arrows?

A

The base of the arrow begins at the original location of the pair of electrons, the barbed head points to their destination; one barb indicates a single electron, two barbs indicates a pair of electrons (what usually occurs)

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4
Q

Which amino acids contribute to the flow of electrons?

A

Polar amino acids, Basic amino acids, and Non-polar amino acids.

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5
Q

How many valence electrons does a nitrogen atom have?

A

3 -> can form 3 sigma bonds, or a sigma and pi bond without requiring excess electrons and the induction of a charge.

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6
Q

What are valence electrons?

A

Electrons that are available for bonding with other electrons.

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7
Q

What is the effect of orbital hybridisation?

A

The valence electrons are all contained with in hybridised orbitals which exist at an energy state between the two.

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8
Q

Where are polar amino acids found?

A

On the exterior of the enzyme and at their active sites.

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9
Q

Where are non polar amino acids found?

A

On the interior of the enzyme and active sites.

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10
Q

Examples of polar amino acids:

A

Cysteine (thiol group), Threonine and Serine (hydroxyl group), Aspartic Acid and Glutamic Acid (carboxyl groups), Asparagine and Glutamine (carboxyamide), Histidine (midazole at pH>=7)

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11
Q

Examples of Basic Amino Acids:

A

Histidine (protonated form), Arginine (guanidium (tri amine), and Lysine (amino group)

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12
Q

What forms the secondary structure of proteins?

A

Hydrogen bonding between carboxyl groups and hydrogen of amine groups -> forming Alpha helices and beta barrels.

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13
Q

What is the Ka equation?

A

Ka = [H+][A-]/HA]

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14
Q

10^(pH-pKa) is equal to what?

A

[A-]/[HA} -> therefore can be used to compare ratio of acid to base.

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15
Q

What is pKa?

A

-log10(Ka)

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16
Q

What factors determine the acidity of an organic compoud?

A

The strength of the Y-H bond, The electronegativity of (Y, greater electronegativity increasing acidity), Factors that stabilies Y-(conjugate base) compated to YH (acid), and the nature of the solvent.

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17
Q

What does a greater pKa signify?

A

Weaker acids

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18
Q

What is generic acid-base catalysis?

A

Where nucleophiles donate electrons to a molecule other than hydrogen. Acid-base catalysis is when a proton is transferred in going to or from a transition state.

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19
Q

What are the properties of the Acid-Base catalysis seen in Ribonuclease A?

A

RNase acts as an endonuclease to cleave single stranded RNA into smaller fragments -> a reaction with 18O showed cleavage of the P-O5’ bond -> RNAse A specifically cuts after the pyrimidine base, indicating specific recognition site, uses water for hydrolysis. This reaction has an intermediate product indicating two reaction steps within the mechanism.

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20
Q

How can kinetic studies be used to indicate the presence of pH sensitive amino acid groups?

A

Measuring the Vmax of a reaction in different pH’s -> peak at 7 suggests Histidine groups.

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21
Q

What are the two key histidine groups within RNase?

A

His12 and His119

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22
Q

What are the roles of the histidine groups within the RNase active site?

A

Studies suggest (bell curve vmax-y and pH-x centred around 7) one acts as a general acid and the other a general base (this role is interchangeable).

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23
Q

What is the role of RNase A?

A

To cleave single-stranded RNA

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24
Q

What is the overall structure of RNase A?

A

A V-shape made up of 3-alpha helices and a 3-stranded, antiparallel beta-sheet, with each strand joined by 4 SS bridges. The active site is sat within the cleft of the V shape, with His119 on the left interior side, and Lys41 and His12 on the N-terminal coil, but in proximity to the other histidine.

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25
Q

What are the active site residues of RNase?

A

His119, His12, and Lys41

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26
Q

Why can’t RNase A target purines?

A

It’s specificity pocket is too small, only allowing binding to pyramdines.

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27
Q

What are the specificity pocket residues of the RNase and what are their purposes??

A

Threonine 45 -> Hydrogen bonding to the amide group of the aromatic ring.
Phenylalanine 120: forms Vdw contacts with the base.
Serine 123 - hydrogen bonding to aromatic amine.

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28
Q

What is Angiogenin:

A

A homologue of ribonuclease A used in the treatment of tumours and aids the health of blood vessels.

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29
Q

Describe the initiating of the RNase A Acid-Base Catalysis reaction mechanism?

A

His12 acts as a base, it donating an electron pair from a conjugate base amine group to the the 2’ hydroxyl group of the ribose ring. An electron pair is then donated from the group’s oxygen to the phosphate, severing it’s bond with the 5’ oxygen; which then attacks the His 119 (acid) amine group, forming a hydroxyl group, attached to the R group/ pyrimidine.

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30
Q

Describe the second (final) step of the RNase A Acid-Base Catalysis reaction mechanism?

A

The deprotonated His119 residue attacks the hydroxyl-pyrimidine molecule formed during the initiation step. The oxygen of the Hydroxy group attacks the phosphate, cleaving the 2’ phosphate bond. The 2’ Oxygen now has an excess electron pair which then attacks the protonated His12 residue, restoring it as a Bronsted Base.

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31
Q

What are isozymes?

A

Enzymes which share function but differ by amino acid sequence, they can be distinguished by differences in optimal ph, kinetic properties, or immunology.

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32
Q

What are proteases?

A

Enzymes that hydrolyse polypeptide chains.

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32
Q

What is a scissile bond?

A

A bond at which hydrolysis cleavage occurs

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33
Q

What is the repeating unit of amino acids (peptide backbone)

A

N, Carbon Alpha (with R group), Carbon with Aldehyde Oxygen

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33
Q

What are the major classes of proteases?

A

Serine Proteases, Thiol/Cysteine Proteases, Metallo Proteases, Aspartyl acid Proteases, Threonine Proteases, Glutamine Proteases.

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34
Q

What are Metallo Proteases?

A

Metallo Proteases use metal ion cores to aid the catalysis of reactions.

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35
Q

What is the function of a specificity pocket?

A

To attach to R groups specific to the target substrate. (increase the specifcity of the enyme and aid arrangement)

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36
Q

What are “hill” regions of enzymes?

A

non-specific binding regions that bind to ketone groups.

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37
Q

What is P1 when counting proteins?

A

P1 is the main specificity site after which cleavage occurs.

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38
Q

How do you number proteins?

A

Upstream of the scissile bond count upwards as you count against the substrate protein direction (p1, P2,…)
Downstream of the scissile bond count upwards as you count the following the substrate protein direction. (P1’, P2’, ….’)

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39
Q

Examples of specificity groups:

A

Phenylalanine (Aromatic, large, hydrophobic), Alanine (very small), Arginine (basic), and Lysine(basic)

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40
Q

Chymotrypsin is an example of what kind of enzyme?

A

A serine protease.

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41
Q

What is the name of the precursor enzyme of Chymotrypsin?

A

Chymotrypsinogen.

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42
Q

What are the target sites of Chymotrypsin?

A

Polypeptide scissile bonds downstream of large aromatic residues.

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43
Q

What is PMSF (testing of chymotrypsin)?

A

PMSF (phenyl methane sulphonyl fluoride) inhibits serine 195, inhibiting the enzyme totally

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44
Q

What is TPRK (testing of chymotrypsin)?

A

Inhibitor of His57, stops enzyme functioning

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45
Q

What is the effect of chain folding on active sites?

A

Chain folding allows for residues which are far apart in the protein sequence to be in proximity of each other.

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46
Q

What 3 residues form the catalytic triad within chymotypsin?

A

Asp102, His57, and Ser195

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47
Q

What is a catalytic triad?

A

When an active site has 3 core catalytic amino acids in proximity which directly interact in the enzymatic action.

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48
Q

How does the catalytic triad of chymotrypsin (serine protease) initiate the reaction?

A

The negatively charged deprotonated aspartic acid residue attracts the hydrogen of the histidine residue, flipping it’s orientation and leaving the nitrogen with the lone pair of electrons to attack and deprotonate the serine residue allowing for it to attack the substrate via nucleophilic addition.

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49
Q

What is an oxyanion hole?

A

The oxyanion hole is a region of the active site where the backbone amide hydrogens of catalytic residues are positioned to point their catalytic groups at the active site.

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50
Q

Chymotrypsin Mechanism Step 1, following the deprotonation of serine:

A

The deprotonated Ser 195 attacks the carbon (bound to the amide group), causing the cleavage of the C=O pi bond; The now negatively charged oxygen (stabilised by the oxyanion hole) donates a pair of electrons to the carbon, then donating a pair of electrons to the bound nitrogen, cleaving their bond. Leaving a acyl enzyme intermediate. The Nitrogen with bound R group then attacks the protonated Histidine’s hydrogen, forming an N-terminal group of the amino acid (product 1).

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51
Q

Chymotrypsin Mechanism Step 2, following the deprotonation of serine:

A

The deprotonated His 57 attacks a water molecule, using a Nitrogen’s lone pair of electrons, which then causes its cleavage, causing an OH molecule to attack the intermediate of nucleophilic addition. Following this the now negatively charged oxygen donates a pair of electrons to the carbon, which then donates a pair of electrons to the Oxygen belonging to the serine, cleaving the intermediate from the enzyme. The negatively charged oxygen then acquires the proton released by the broken down water molecule.

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52
Q

When is histidine 57 protonated in chymotrypsin’s reaction mechanism?

A

During initiation and and deprotonation of serine 195.

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53
Q

What type of catalysis is the chymotrypsin mechanism?

A

General Acid-Base catalysis

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54
Q

What are the three groups of Serine protease we are taught about?

A

Chymotrypsin, Elastase, and Trypsin

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55
Q

What differentiates which substrates can be catalysed by the different serine proteases?

A

The residues at the specificity sites of these serine proteases.

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56
Q

What substrates are targeted by Chymotrypsin?

A

Uncharged molecules

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57
Q

What substrates are targeted by Elastase?

A

Small uncharged molecules -> because of V226, and T216 decreasing the size of its specificity pocket.

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58
Q

What substrates are targeted by Trypsin?

A

positively charged molecules, because its specificity pocket has a D189 residue (aspartate), which is negatively charged.

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59
Q

How are polypeptide substrates correctly orientated for Acid-Base catalysis by serine proteases?

A

The substrates main chain oxygen (proton acceptor) and nitrogen (proton donor) are used in hydrogen bonding with the proteases backbone.

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60
Q

What is the effect of increasing the number of specificity pockets of an enzyme?

A

Increasing specificity to a particular substrate, limiting the number of target substrates.

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61
Q

What are the characteristics of serine proteases?

A

2 beta barrels, each forming a respective “top” and “bottom” domain, each forming a major helices. They have an alpha helices group and catalytic triad.

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62
Q

For what specific purpose is the oxyanion hole useful for during acid-base catalysis in serine proteases?

A

The cleavage of the tetrahedral intermediate.

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63
Q
A
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64
Q

What is a histag?

A

A tag of 6 histidine residues added to the beginning and end of a protein (N-terminus and C-terminus) used to separate protein via nickel column separation

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65
Q

What is enzyme activation by proteolysis?

A

Enzymes, such as chymotrypsin, are synthesised in a precursor form known as proenzymes (zymogens) -> proteases will then remove a portion/region of this, activating the enzyme (holoenzyme).

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66
Q

What happens to polypeptide fragments which are cleaved from the backbone?

A

Disulphide bonds (tertiary structure) interactions will keep the fragments bound, despite interruptions to the main chain sequence, allowing for greater flexibility.

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67
Q

How does the cleavage of Chymotrypsinogen (zymogen) lead to the activation of Chymotrypsin’s active site?

A

NH3+ group on the N-terminal ile 16 pairs to the Asp194 side chain, altering the confirmations of mainchain residues Gly 193 (correct formation of the oxyanion hole) and Ser 195 (for correct geometry in the in the catalytic triad)

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68
Q

Structure of Proteasome:

A

Multi-subunit cylindrical complex with an interior cave containing a proteolytically active site, mechanistically belonging to the N-terminal. The core is made up on 28 subunits arranged in 4 stacked rings with 7-fold symmetry.

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69
Q

Threonine Protease mechanism: Step 1 formation of tetrahedral intermediate

A

Lys 33 Nitrogen’s lone pair of electrons attacks Thr 1’s (threonine) hydroxyl group, the oxygen then attacks the carbonyl group on the substrate by nucleophilic addition.

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70
Q

Threonine Protease mechanism: Step 2 formation of product 1

A

The negatively charged Oxygen with an excess pair of electrons from the cleavage of their pi bond with the substrate carbon, donates the pair of electrons to the carbon, which then donates it to the adjacent substrate amide group, which then cleaves its scissile bond with the carbon by attacking the positively charge amine of Thr 1, sequestering a proton and leading to cleavage.

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71
Q

Threonine Protease mechanism: Step 3 Attack by water

A

The now deprotonated Thr 1 amine group attacks a water molecule to restore it’s charge, the remaining OH- then attacks carbonyl bound carbon by nucleophilic addition to form a tetragonal intermediate.

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72
Q

Threonine Protease mechanism: Step 4 Release of Product 2 and restoration of catalyst.

A

The now negatively charge Oxygen from the carbonyl group uses their excess pair of electrons to attack the carbon, which then attacks the bound enzyme’s oxygen (cleaving the product from the enzyme), which then attacks the excess hydrogen group originally sequestered by Lysine 133.

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73
Q

What is the key difference in the mechanism of cysteine proteases and serine proteases?

A

Cysteine groups have a thiol group instead of a hydroxyl group. The thiol group more readily acts as a proton donor, because the sulphur is less electronegative, and-so more readily/ more strongly reacts.

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74
Q

What is the general function of cysteine proteases?

A

Bulk degradation in cell processes suchas apoptosis, parasitic infection, and virus maturation.

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75
Q

What are the catalytic residues in a cysteine protease’s active site?

A

Cysteine and Histidine.

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76
Q

What is the pKa of Ser and Cys?

A

Ser Pka = 13, Cys Pka = 8.3.

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77
Q

Cysteine Protease Mechanism: Formation of product 1

A

The histidine residue’s
basic nitrogen with a lone pair donates the lone pair to bind to Cysteine’s thiol group Hydrogen; The now negatively charged sulphur attacks the carbonyl bound carbon the substrate via nucleophilic addition. The now negatively charged oxygen on the substrate donates a pair of electrons to its bond with the carbon to restore its pi bond, causing the C-N bond to cleave by donating a pair of electrons to the nitrogen, the negatively charged nitrogen then attacks the protonated histidine group (forming product 1) and accepts its excess proton.

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78
Q

Cysteine Protease Mechanism: Formation of product 2

A

Water enters the system and is attacked by the basic nitrogen of the histidine, causing the OH- to attack the remaining substrate by nucleophilic addition, the negatively charged oxygen then attacks the carbon, however the C-S bond is now cleaved, due to the the other bonds being stronger. The sulphur group now has an excess pair of electrons which is then used to attack the protonated histidine and restore the enzyme.

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79
Q

What are the similarities between deamidation and peptide hydrolysis?

A

Both use enzymes which use a His/Cys diad to promote hydrolysis of a carbon-nitrogen sigma bond and both have similar arrangements with their carbon, oxygen, and nitrogen.

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80
Q

What is the key difference between deamidation and peptide hydrolysis?

A

In deamidation a terminal amine group is cleaved to form a carboxylic acid, whereas in hydrolysis two amino acids are formed.

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81
Q

What are deamidation enzymes often used for by microorganisms?

A

They’re often used by pathogenic bacteria as toxins.

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82
Q

What’s a key example of a pathogenic bacteria which uses deamidation enzymes to harm humans?

A

Burkholderia psuedomallei -> found in many third world countries -> responsible for “whitmore’s disese” -> similar symptoms to TB so often undiagnosed.

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83
Q

What is a method which can be used to increase the resolution of protein analysis?

A

Once you’ve separated proteins by gel electrophoresis, proceed to rotate the gel by 90’ and then separate it again.

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84
Q

Why can proteomic analysis between pathogenic and non-pathogenic bacteria of the same species be useful?

A

Differences in the proteins expressed between the two can provide insight into potential future drug targets.

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85
Q

Give an example of a protein that is a glutamine deamidase?

A

CNF1, or BPSL1549

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86
Q

What does BPSL15449 target?

A

elF4A (helicase), preventing protein transcription -. leads to Death.

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87
Q

What are optical isomers?

A

Non-super imposable structures with the same molecular and structural formula.

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88
Q

What are the characteristics of Acid-Base catalysis?

A

A metal ion holds residues in place to form structures similar to the oxyanion hole, helping to interact with certain parts of the molecule as it changes position, aiding the collapse of electrons falling back (the 2nd step of catalysis following nucleophilic addition)

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89
Q

What structural characteristics are common amongst Metallo Enzymes?

A

a c-terminus barrel domain and n-terminus caping domain.

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90
Q

What are the two subgroups of the enolase superfamily?

A

MLE and Mr groups.

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91
Q

How do the two enolase subgroups differ?

A

They differ by the residue which carries out the catalysis of their reaction, either using a Lys diad (MLE) or Lys, His, and Asp (MR); they also differ in the specific resiudes used to hold the metal ion.

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92
Q

What is the structural formula of an enolate anion?

A

CH3CO=CH2

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93
Q

Why do enolate anions form feasibly, despite the unfavourable loss of a C-H bond?

A

The hydrogen adjacent to the carbonyl group is acidic, donating its pair of electrons to the bound carbon when attacked by a base (metal ion).

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94
Q

What are the two steps of enolase action?

A

Step 1: Deprotonation by base and transfer of donated pair of electrons from carbon, to carbonyl’s oxygen of carboxylic acid.
Step 2: This process reverses, but because of the strain of the the remaining hydroxyl group is pulled backwards, allowing for the newly bound proton to be brought forward, forming an optical isomer of the original substrate.

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95
Q

Metal Ion catalysis mechanism of Mandelate Racemase:

A

Lysine residue donates a pair of electrons to sequester the substrate’s acid-like hydrogen -> this electron pair is then donated to the carbonyl oxygen. The negative oxygen formed is held in place by glutamate (via van der waals) and the charge collapses in on itself, cleaving the C=C, causing the restoration of the carbonyl group and the other carbon then recruits a proton from the histidine.

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96
Q

What is the function of the enolase super family?

A

To switch the chirality
of a substrate (usually switching the chirality of groups on carbon adjacent to a carbonyl.

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97
Q

What ions are used to oxidise/ reduce species?

A

Hydride ions.

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98
Q

What is the molecule routinely used to source hydride ions?

A

BH4 (borohydride)

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99
Q

Why isn’t BH4 used for reduction/oxidation in biological systems?

A

BH4 is very effective, however cannot be used very selectively leading to death, NAD(P)H families of cofactors are used instead as they can be used much more selectively.

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100
Q

What is the structure of NAD(P):

A

A nicotinamide mononucleotide, phosphate linker, and Adenosine base.

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101
Q

What is significant about the difference between NAD(P) and NAD(P)H?

A

In the oxidised form, the nicotinamide ring is positively charged with three double bonds, whereas when reduced one of the double bonds is lost, the positive charge is lost, and the ring is no longer planar. The C4 position is out of the plane, with two attached hydrogen groups.

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102
Q

What are the sources of nicotinamide rings?

A

Nicotinic acid, tryptophan degradation, NAD reuse/ recycling

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102
Q

How does NAD(P)H reduce carbonyl groups?

A

Nucleophilic attack of a hydride ion (from the nicotinamide ring), this frees a pair of electrons for the carbonyl’s oxygen to attack an acid and sequester a hydrogen.

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103
Q

Why is the positioning of Hydride Transfer very critical?

A

Hydride Transfer Reactions are very highly stereospecific, the distance and angle at which they occur is critical (3~ 3.5~ Angstroms and 107’) -> this ensures the correct transfer of the hydride.

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103
Q

What are the two systems of Fatty Acid Synthesis?

A

FAS Type 1: Catalytic Domains of 1 or 2 polypeptides - found in vertebrates
FAS Type 2: In plants and bacteria, multiple discrete polypeptides catalysing each individual enzymatic step.

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103
Q

What is the source of the hydrogen which reduces the NAD(P)H cofactor?

A

From the enzyme (however this is then restored via other means)

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104
Q

What is the result of the fatty acid elongation cycle?

A

Each cycle adds 2 extra carbon units to the carbon chain.

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105
Q

What system of fatty acid synthesis is a common target of drugs?

A

FAS Type 2

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106
Q

What is ACP?

A

ACP is an Acyl Carrier Protein which allows for the cell to solubilise hydrophobic chains

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107
Q

How does the state of ACP differ between FAS 1 and FAS 2?

A

In FAS 1 ACP is associated with large machinery; whereas in FAS 2 ACP is separate and freely floating around as it carries the growing fatty acid chain on a fishing-rod like structure until it finds the target enzyme.

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108
Q
A
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109
Q

Describe the mechanism of beta-ketoacyl reductase:

A

Uses NAD(P)H to reduce a ketone group into a hydroxyl group.

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110
Q

What is the role of enoyl reductase?

A

Reduces the double C=C formed by dehydratase in FAS

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111
Q

Describe the ACP structure:

A

The ACP is made up of the transport protein and links to the acyl group via a serine residue; this residue is tucked inside the protein which therefore means the chain is tucked inside, only released and arranged upon reaching the active site of the next enzyme in the cycle.

112
Q

What is the function of Beta Keto ACP reductase (BKR)?

A

Catalyses first reductive step of acid elongation cycle

113
Q

What is the general structure of BKR (excluding active site)?

A

BKR is tetrameric made of 4 identical alpha helical polypeptide chains with loop regions, each with an active site (theoretically 4 reactions could occur simultaneously.), surrounding a central beta sheet. This forms a Rossman Fold structure with 2-fold symmetry.

114
Q

What is the significance of BKR loop regions?

A

They determine BKR’s enzyme specificity and their ability to interact with other copies of the polypeptide.

115
Q

BKR Active Site:

A

contains a conserved tyrosine (and a conserved lysine) near the nicotinamide ring of the NAD cofactor

116
Q

How does the NAD cofactor sit in the BKR during fatty acid synthesis?

A

the phosphate groups sits at the positive end of a pair of alpha helices (dipolar) and the adenine ring sits within a pocket on the protein surface

117
Q

BKR reaction mechanism:

A

The NADPH donates a hydride to the C3 of the acyl chain, because the carbon runs out of valence electrons the C-O pi bond is broken, the now negatively charged Oxygen acquires the hydrogen of the tyrosine hydroxyl group.

118
Q

What is the role of the lysine residue in BKR?

A

The lysine stabilises the intermediate (with the negatively charged oxygen), allowing for the acquisition of the hydrogen from the tyrosine.

119
Q

What is the ENR mechansim?

A

Hydride is transferred from NADH to C3, this rearranges the substrate to form an enolate anion intermediate; the carbonyl oxygen acquires the tyrosine’s hydrogen to form a hydroxyl group. Tautomerisation then occurs in which the product transfers between enolase form and ketone form.

120
Q

Describe the tautomerisation mechanism in ENR:

A

The Enolase intermediate with a C1 hydroxyl group -> Ketone form (desired):
C1/C2 double bond donates a pair of electrons to the hydrogen of the hydroxyl group (acquiring it), the now negatively charged Oxygen has the valence to then form a pi bond with C1.

121
Q

Compare BKR and ENR:

A

They show a high degree of structural similarity, but low sequence similarity.
Active site critical tyrosine (highly maintained) residues do not directly superimpose but the phenyl hydroxyl groups are very close in position.

122
Q

Describe the structure of ENR:

A

Tetramer, with a Rossman fold; their monomer looks similar to other members within its family.

123
Q

What is the key difference between BKR and ENR?

A

The distance between the tyrosine and lysine residues is double that of BKR (4 residues) in ENR (8 residues). This is beacause the ENR hydride is donated to the 1’ ketone group, whereas BKR’s is donated to the 3’ ketone.

124
Q

Why is Diazaborine not an appropriate medication to treat pathogenic bacterial infection?

A

The main issue is that boron is very toxic and would cause brain damage, however an alternative reaction mechanism not using boron was never found to be as effective.
Additionally exposed bacteria very quickly developed resistant mutants (simple glycine to alanine)

125
Q

How does Diazaborine inhibit ENR?

A

Diazaborine sits atop the Nicotinamide ring of the NAD cofactor, forming a covalent link with its boron hydroxyl group and the ribose sugar of the NAD -> blocking the substrate -> outcompeting the substrate and preventing hydride transfer.

126
Q

How does Triclosan inhibit ENR?

A

triclosan has a structure mimicking the enolate intermediate of the ENR pathway (its oxygen and dichlorobenzene mimicking the ACP); the triclosan then undergoes van der waals.

127
Q

What are the two drugs developed to target ENR?

A

Triclosan and Diazaborine

128
Q

Why is triclosan more effective than diazaborine as drug targeting the ENR?

A

Triclosan is much less toxic (only having side effects at excess doses) and it’s much more difficult for bacteria to develop resistance.

129
Q

What are the two types of Β-hydroxyacyl dehytrases in bacteria?

A

FAB A and FabZ.

130
Q

What is the structure of Β-hydroxyacyl dehytrases?

A

A dimer made up of 2 monomers which have an underlying fold, the “hotdog” fold; an alpha helix, wrapped in beta sheet. There’s a highly conserved histidine, glutamate, and aspartate (glutamate and aspartate both carrying amino acids) conserved within both monomers

131
Q

What is the benefit of dimerisation of Β-hydroxyacyl dehytrases?

A

Allow for the functional groups to be in proximity with each other, whereas in their monomer form the residues would be too far apart.

132
Q

What is the key difference between Fab Z and Fab A?

A

Fab Z uses a glutamine residue to donate a hydrogen to enable the C3 hydroxyl group to leave as a water molecule, whereas Fab A uses an Aspartic Acid group.

133
Q

Describe the Step 1 of the mechanism of Β-hydroxyacyl dehytrases:

A

The deprotonate nitrogen of the histidine residue attacks a C2 proton using its pair of electrons, this transfer of electrons then targets the C2-C1 bond to form a double bond; Because C1 is at its maximum valence and-so severs it’s C-O pi bond, inducing a negative charge on the Oxygen.

134
Q

Describe the Step 2 of the mechanism of Β-hydroxyacyl dehytrases:

A

The negatively charged oxygen transfers a pair of electrons to its C-O bond, cleaving the C1-C2 pi-bond, transferring the electrons to the C2-C3 bond, causing C3 to cleave its C3-O sigma bond, and the OH- attacks the Glutamine/Aspartic Acid, recruiting a proton and leaving the system as H2O.

135
Q

What is the importance of phosphodiester bonds in DNA?

A

They’re critical for holding nucleic acid bases together and doesn’t cleave naturally on its own (however can be broken down during DNA processing)

136
Q

What is the associative scheme for the hydrolysis of nucleic acids?

A

The Nucleophile (OH) attacks the phosphate from the opposite side of the desired leaving group, this cleads to the cleavage of the ester bond, in which the cleaved oxygen (with attached R group) then attacks a H2O molecule to acquire a proton, regenerating an OH- ion.

137
Q

How many valence electrons does phosphorous have?

A

5

138
Q

What are the two mechanisms of phosphate hydrolysis?

A

The associative and dissociative mechanisms.

139
Q

What occurs during the dissociative hydrolysis of DNA:

A

The leaving group is cleaved before the nucleophilic attack of water, simultaneously losing a proton to generate a nucleophile, attacking phosphorous, and leaving the R group deattached.

140
Q

Why can RNA hydrolyse itself?

A

RNA has an extra C2’ hydroxyl group which can be the source of an attacking nucleophile.

141
Q

What are the products of RNA phosphodiester hydrolysis?

A

A random mixture of RNA molecules with either a 3’ hydroxyl and 2’ phosphate, or 3’ phosphate and 2’ hydroxyl.

142
Q

RNA self hydrolysis mechanism:

A

A base attacks the 2’/3’ hydroxyl group, acquiring a proton and generating a nucleophile oxygen (negatively charged) which attacks the phosphate, forming an intermediate by nucleophilic addition to the phosphate. The target phosphoester bond then attacks the protonated base, cleaving it the two RNA nucleotides. These product with the2’ 3’ phosphoester bonds is then resolved by hydrolysis by water, forming either a 3’ or 2’ phosphate product.

143
Q

What are the key residues in the RNAse A active site?

A

Histidine 12, Lysine 41, Histidine 119

144
Q

What is the first step of the RNAse A reaction mechanism?

A

H12 aquires the hydrogen from the C2’ hydroxyl group generating a nucleophile, which attacks the phosphate by nucleophilic addition; The C5’ ester bond is then cleaved by donation of a pair of electrons to H119 to acquire the proton (to form a hydroxyl group)

145
Q

What is the difference between RNA hydrolysis and DNA hydrolysis?

A

DNA doesn’t have RNA’s additional hydroxyl group, therefore a general base, and general acid for the catalysis of the reaction.

146
Q

What examples of Type II restriction Endonucleases do we use as examples?

A

EcoRI and EcoRV

147
Q

What is the general structure of Type II restriction endonucleases?

A

Dimeric enzymes with a central beta sheet flanked by alpha helices, forming a cup shape which can adhere to and scan along the DNA.

148
Q

Where do Restriction Endonuclease enzymes tend to bind?

A

16/18 base pair long palindromic sequences.

149
Q

What is the purpose of function side chains in the recognition of palindromic DNA sequences by Restriction Endonucleases?

A

The side chains detects slight distortions caused by variations in the DNA backbone, forming van der waals and hydrogen bonds to ensure the correct recognition of the sequence.

150
Q

What is the conserved motif in Type II restriction endonucleases?

A

a proline followed by an aspartate then by either an aspartate or glutamate, a random amino acid and then lysine

151
Q

Why do REase use metal ions?

A

Metal ions are used to aid the deprotonation step of the species attacking the water; however they don’t remove the proton, but instead stabilise the resultant OH- ion. They also may be used to enable the negative charge on the pentavalent intermediate, and may stabilise the leaving oxyanion group when the metal ions are in an abundance.

152
Q

What metals are used by REases?

A

Nickel, Cobalt, Zinc, (primarily) Magnesium.

153
Q

What is MVAI?

A

A restriction endonuclease that has a water molecule positioned by the Lysine residue -> this will make an inline attack on the substrate’s (DNA ) phosphate upon the formation of the hydroxide ion.

154
Q

Why are metal ions useful in active site arrangement (structural function)?

A

Metal ions have coordination geometry requirements, ensuring species are fixed in location

155
Q

What are the two REase mechanism of action?

A

The one metal ion and two metal ion mechanisms

156
Q

What is the significance of the Rossman fold?

A

It’s important for the substrate binding of the phosphate.

157
Q

REase one metal ion mechanism:

A

“Substrate-assisted cleavage” - The phosphate’s negatively charged oxygen attacks an incoming water molecule, which then forms a hydroxide which then attacks the phosphate with the 3’ phosphoester bond desired for cleavage.

158
Q

What is the role of Mg2+ in REase one metal ion mechanism?
(Not NHN)

A

The Mg2+ ion stabilises the intermediate formed, but also holds residues in place for the correct arrangement for the reaction take place.

159
Q

Why is the stabilisation of products very delicately controlled (enzyme/product interaction)

A

A level of instability is required for the dissociation of the product, allowing for the enzyme to interact with more substrate.

160
Q

REase two metal ion mechanism:

A

A conjugate base Lys/Glu attacks a H2O molecule, forming a hydroxide ion which attacks the phosphate group by nucleophilic substitution; the negative charge generated on the oxygen collapses, cleaving the 3’ phosphoester bond; A water molecule’s proton is donated to generate negative water on the cleaved nucleotide (3’).

161
Q

What is the role of the Mg2+ cores in two metal ion REases?

A

Mg1 - facilitates the deprotonation of a water molecule by nearby basic residue, helping to position the generated nucleophile.
Mg2 - interacts wiht the leaving oxyanion and may position a water molecule for proton donation.
The two metal cores are 4 Angstroms apart, stabilising the negative charge on the targeted pentavalent phosphorous.

162
Q

What are NHN nucleases?

A

A class of nuclease enzyme using histidine, aspartate, and asparagine, using the two latter to stabilise their metal ions for arrangement and the histidine for as a conjugate base. This mechanism/domain is found in Cas9 of the CRISPR system.

163
Q

What are the 3 components of a DNA monomer (nucleotide):

A

A pentose sugar (deoxyribose) with 3’ hydroxyl group, a phosphate, and a base.

164
Q

What are the 4 DNA bases?

A

Adenine and Guanine (purines), Thymine and cytosine (pyrimidines)

165
Q

What links nucleotides together?

A

Phosphodiester linkages between the 3’ hydroxyl and the C5’ phosphate of the adjacent nucleotide.

166
Q

What determines the primary structure of a protein?

A

The order of amino acids, determined by the encoding codons.

167
Q

What force overcomes the reduction in entropy caused by the formation of DNA?

A

Hydrogen bonding between DNA bases, makes delta G negative and thus thermodynamically feasible.

168
Q

What is meant by DNA replication being semi-conservative?

A

New DNA strands are formed from the parent strands, however they themselves are also incorporated into the new DNA molecules.

169
Q

Why is the accurate replication of DNA important?

A

Malfunctioning can lead to mitotic catastrophe (e.g. cancer/tumorigenesis) and cell death.

170
Q

What molecule is released upon the addition of a nucleotide during DNA replication?

A

A pyrophosphate.

171
Q

What is the general reaction of DNA replication?

A

(DNA)n + dNTP <–> (DNA)n+1 + PPi

172
Q

What kind of reaction is DNA replication?

A

Substitution Nucleophilic Bimolecular Reaction.

173
Q

What enzyme catalyses DNA replication?

A

DNA polymerase.

174
Q

Describe the mechanism by which DNA polymerase catalyses DNA replication?

A

3’ hydroxyl is deprotonated and nucleophilic attacks the alpha phosphate, causing electrons to be transferred leading to the breakage of the bond between the beta-gamma phosphate and the 1st oxygen; this leads to the formation of an ester bond between the 3’ OH and the alpha phosphate.

175
Q

Why is it significant that DNA polymerase possesses multiple domains?

A

DNA polymerase can form opposite reactions, its upper “hand” region performs polymerisation, whereas its 3’ to 5’ exonuclease domain can remove nucleotides.

176
Q

How does DNA polymerase switch between its functions?

A

Changes in protein conformation in reaction to changes in the DNA backbone.

177
Q

What method did Scientists use to observe the active site of DNA polymerase?

A

They provided the enzyme with a primer lacking the 3’ hydroxyl group (dideoxy-terminate primer), locking the enzyme in its substrate binding conformation, allowing for observation using X-ray crystallography.

178
Q

What are species found in the DNA polymerase active site?

A

2 Aspartic acid residues and a Mg2+ ion core

179
Q

What mechanism is the mechanism at the active site of exonuclease similar to?

A

The two metal ion mechanism of polymerase 9but reversed)

180
Q

Outline the mechanism of the exonuclease site of DNA polymerase?

A

A tyrosine residue acts as a conjugate base, attacking the a water molecule, stabilised by Glutamine , which is then cleaved in to a hydroxide ion; this hydroxide ion then attacks the phosphate group by nucleophilic substitution, cleaving the phosphate’s bond to the nucleotide opposite to the side of the phosphate targeted.

181
Q

Why is the specificity of DNA polymerase E7 important?

A

The polymerase can only polymerase correct/bonafede nucleotides, which prevents the polymerisation of damaged DNA.

182
Q

What is the role of trans-lesion synthesis polymerases?

A

To ensure replication is uninterrupted.

183
Q

How is activity between Specific Polymerases and Trans-lesion Polymerases mediated?

A

Either by processivity factor (proteins that actively switch between the two) or the dissociation of the enzymes at different point along the DNA.

184
Q

What is the primary enzyme for DNA synthesis?

A

DNA polymerase III, because it’s fast and accurate.

185
Q

Which DNA polymerases can bypass lesions?

A

II, IV, and V can bypass lesions and continue replication when the replication machinery encounters damaged DNA.

186
Q

What is the purpose of having multiple types of DNA polymerase?

A

DNA synthesis must be very accurate, and-so there must be a range of different polymerases to ensure accurate replication, whilst also minimising disturbance caused by mutations and damage; all of which couldn’t be fulfilled by a single more general enzyme.

187
Q

Which type of DNA polymerase is used in DNA repair in emergencies and is “error prone”?

A

DNA polymerase II

188
Q

What is unique about polymerase enzymes compared to others?

A

Polymerase doesn’t dissociate from the DNA substrate, instead moving along and further increasing the chain length.

189
Q

What is the O-helix on DNA-polymerase?

A

An alpha helix on the finger domain is joined to the O1-helix by a short linker loop; it allows for the DNA to move backwards relative to the polymerase, bringing 5’ nucleotides to the insertion site.

190
Q

What evidence supports the role of the O-helix/O1-helix in DNA polymerase activity?

A

The structure is conserved in polymerases between species.

191
Q

What is the benefit of the slower speed of polymerisation of DNA polymerase II?

A

The error prone polymerase is slower to reduce the capacity for errors that can be caused by their less specific binding.

192
Q

Why can’t the 2nd metal ion be observed in X-ray crystallography structures retrieved from DNA polymerase inhibited by Dideoxy-terminate primers?

A

The metal ion will no longer be held in place due to the lack of 3’ hydroxyl group.

193
Q

What are the initial stages of protein purification?

A

Cell harvesting (through centrifugation) and Cell Lysis by mechanical or non-mechanical methods.

194
Q

What are the mechanical methods of Cell lysis?

A

Sonication

195
Q

What are the non-mechanical methods of Cell Lysis?

A

Osmotic Shock

196
Q

What are parameters by which proteins can be distinguished/separated?

A

Solubility (hydrophobicity), Size (size exclusion/gel filtration), Charge (ion exchange), and Affinity (Affinity Chromatography)

197
Q

Gel Filtration Method:

A

A column is filled with tiny beads, containing microscopic channels (size varies/ controlled), smaller proteins will be traverse these channels, whereas larger proteins will be washed away more readily by buffer; therefore the smaller proteins travel down the column more slowly and large proteins will be the first detected to leave the column.

198
Q

Affinity Chromatography Method:

A

The binding molecules are attached to the dye/cofactor by linker arms, meaning when the protein is poured down the desired protein will bind to the column, and the unwanted proteins will wash away by buffer. The desired proteins are then eluted using by free ligand or salt buffer.

199
Q

What are the commonly used affinity ligands?

A

o Partner molecules (inhibitors, cofactors, or other proteins)
o Specific antibodies
o Dyes (pseudo affinity)
o Glutathionine (binds to GST – Gluthionine S Transferase)
o Ni2+ (binds to poly histidine, e.g. 6x His-tags -> not found often naturally and-so can be used as an effective filtration method.)

200
Q

When is Gel Filtration Generally used?

A

As a size selective method its often used to polish samples as the last step, however can be used earlier in the protocol.

201
Q

What is the benefit of using a dye column over a cofactor column in affinity chromatography?

A

Dye columns are more robust and cheaper to build.

202
Q

How is each step of a protein purification process evaluated?

A

SDS-PAGE is used to identify changes to bands presence (changes to their fluorescence) to determine the amount and purity of the extract. An activity assay is then performed to observe whether or not the extracted protein is unfolded (unfolded) or folded (operational)

203
Q

Why is each step of a protein purification protocol evaluated?

A

To determine the effectiveness of individual steps further streamline the process and cut unnecessary steps.

204
Q

What are the 3 final steps of evaluative methods of Protein Purification?

A

Specific Activity Calculations, Percentage Yield, and Purification Factor.

205
Q

What are the basic principles of SDS-PAFE?

A

The protein mixture is denatured with heat and its charge density is made uniform using the anionic detergent SDS -> this sample is then loaded onto a gel and an electric field is applied, with the proteins travelling towards the Cathode (+ve); smaller proteins travel faster and further through the gel, separating the proteins by size.

206
Q

What is the gel used in SDS-PAGE?

A

Polyacrylamide gel

207
Q

What is the benefit of SDS-PAGE as an evaluative step during protein purification?

A

SDS-PAGE reveals how clean the target protein is from other proteins.

208
Q

What dye was used for affinity chromatography with GDH?

A

Remazol red dye.

209
Q

What is an easy optical activity assay which can be performed following dye affinity chromatography?

A

Spectrophotometry -> measuring the absorbance of successive elutions.

210
Q

How is activity measured during spectrophotometry:

A

The dyed protein will undergo a different conformation upon binding to substrate, this change can cause the absorbance/fluorescence to increase/decrease. The rate at which this changes over time can be using repeated measurements; A line of best fit can then be derived providing an estimation of the initial rate.

211
Q

What are the units of Enzyme activity?

A

units/ml

212
Q

What is a unit? (In terms of enzyme activity?)

A

1 unit of activity is the amount of enzyme needed to convert 1 micromole of substrate to product in 1 minute.

213
Q

What is the equation for Specific Activity?

A

Specific Activity is Enzymatic activity / Protein Concentration.

214
Q

What is Specific Activity?

A

Specific Activity is a measure of purity of an enzyme preparation

215
Q

What are the units of enzyme activity “units”?

A

micromoles / minutes

216
Q

How do you calculate Purification Factor:

A

specific activity after purification / specific activity before

217
Q

Absorbance:

A
218
Q

What is the name of the time-tested colorimetric assay we used to measure the protein activity and purity of our GDH?

A

Bradford Assay.

219
Q

What is the effect of an enzyme on the rate of reaction?

A

They increase the rate of reaction by making reactions more thermodynamically and kinetically feasible.

220
Q

In an irreversible unimolecular reaction: How do you calculate the probability A will transform into B?

A

P(A->B) = K1 x t

221
Q

In an irreversible unimolecular reaction: What is K1?

A

The rate constant

222
Q

In an irreversible unimolecular reaction: How do you calculate the rate of change in concentration of A?

A

Rate of Change = V,

V = d[A]/dt = -k1x [A]

223
Q

In an irreversible unimolecular reaction: What are the units of v?

A

micromole /s

224
Q

In an irreversible unimolecular reaction: How do you calculate the rate of change in concentration of B?

A

v = d[B]/d[t] = k1[A]

225
Q

What is the numerical approach for determining the half life of a unimolecular irreversible reaction?

A

Measure the concentration of the substrate over the duration of the reaction (continuous direct); the rate of change in [A] can be determined at each time point. The half-life can be found by plotting a graph of [A] / microM over time, and finding the time at which [A] has halved.

226
Q

What is the equation relating substrate concentration, rate constant, and time ; for unimolecular irreversible reactions?

A

[A] = [A]0 x e^(-kt)

or

ln([A]/[A]0) = -kt

227
Q

When are linear form graphs appropriate (via the use of logarithms)?

A

When plotting information which deals with multiple exponential values that make the unaltered values much more difficult to observe.

228
Q

How do you calculate the concentration of [B] given the [A]0, rate constant, and time elapsed?

A

[B] = [A]0 x (1-e^(-kt))
(The graph forms an inverse curve to the alternative [A] = {A]0 x e^-(kt))

229
Q

How do you derive the equation: [B] = [A]0 x (1-e^(-kt))

A

[B] = [A]0 - [A]
[B] = [A]0 - [A]0 x e^(-kt
[B] =[A]0 x (1 - e^-(kt))

230
Q

In a unimolecular reversible reaction: What is the rate of change of A equation? (numerical approach)

A

d[A]/[dt] = -k+1 [A] + ki-1[B]

231
Q

In a unimolecular reversible reaction: What is the equation to calculate the average time the species is in state A?

A

A = 1/k1

232
Q

In a unimolecular reversible reaction: What is the equation to calculate the average time the species is in state B?

A

B = 1/k-1

233
Q

In a unimolecular reversible reaction: What does the average time in a state tell you?

A

The average time in the state over the entire population of the substrate/reactant, rather than an individual molecule.

234
Q

What is occurring at equilibirum?

A

The reaction is still ongoing, however no net change occurs in the species as the two rates are completely balanced.

235
Q

In a unimolecular reversible reaction: What is the equation to calculate the equilibrium concentration of A?

A

[A]eq = 1/(1+k) x total conc.

236
Q

In a unimolecular reversible reaction: What is the equation to calculate the equilibrium concentration of B?

A

[B]eq = K/1=K

237
Q

In a unimolecular reversible reaction: What is Kobs?

A

Kobs is the observed constant:
Kobs = K+1 + K-1

238
Q

In a unimolecular reversible reaction: What is the equation to calculate the concentration of [A] at a given time?

A

[A] = [A]0^(e(k+1+k1)t + B

239
Q

In a unimolecular reversible reaction: What equation links equilibrium concentrations of A and B with the rate constants of the forwards and backwards reaction?

A

K-1x [B]eq = K+1 x [A]eq

240
Q

In a unimolecular reversible reaction: When the product and substrate are in equilibrium what is the ratio of proportion between the two?

A

[A] : [B]
1 : Keq

This is derived from the equation Keq = [B]/[A]

241
Q

In bimolecular irreversible reactions: what are the simplifications in lab which can be used to calculate/estimate the time dependency of reactions?

A

The concentrations [A] = [B] or [A] &laquo_space;[B] -> allows for pseudo-first order, which makes the changing concentrations simple and trackable.

242
Q

In bimolecular irreversible reactions: What is the equation to calculate the concentration of [A] at a given time?

A

[A] = [A]0 x e^(-kobs x t)

243
Q

In bimolecular irreversible reactions: What is the equation for Kobs?

A

Kobs = K+1 x [B]

244
Q

Why are pseudo first order results not comparable to real first order results?

A

They have different units.

245
Q

What is meant when the y-axis is labelled [A]/[A]0?

A

The y-axis shows the proportion of [A] remaining in the reaction mixture. (Starting the reaction at 1)

246
Q

What is useful about the Pseudo-first order approach to bimolecular irreversible reactions:

A

Pseudo-first order kinetics has very broad use, giving useful insights into two step mechanisms. (e.g. they can help to deduce rate constants by plotting results) ~ numerical method

247
Q

What is the rate of change of [A] in a bimolecular reversible reaction?

A

d[A]/dt = -k+1[A][B] + K-1[C]

248
Q

In a bimolecular reversible reaction what relationship can be derived from the rate of change of [A] equation?

A

At equilibrium d[A]/dt = 0:

k+1[A][B] = k-1[C]

249
Q

What is Kd?

A

Kd is the dissociation constant, a measure of binding strength. A smaller Kd indicates tighter binding.

250
Q

In a bimolecular reversible reaction: What is the equation of Kd?

A

[A][B]/[C]

251
Q

At Equilibrium in a bimolecular reversible reaction: What is the equation linking concentration, rate constants, and Kd?

A

Kd = [A][B]/[C] = K-1/K+1

252
Q

Because signals are often measured instead of ligand concentration what is done to estimate/measure rate?

A

The total signal change (when distinct between bound and unbound form) is measured as a function of concentration, being mapped as a percentage of binding to the proportion of total ligand.

253
Q

In a protein ligand interaction: What is the equation to find the fraction of bound protein?

A

[PL]/Pt

254
Q

In a protein ligand interaction: What is the equation linking concentrations and Kd?

A

[PL] = [P][L]/Kd

255
Q

In a protein ligand interaction: How can Kd be found from a Protein/ligand binding curve?

A

Finding the [L] when binding is at 0.5

256
Q

In a protein ligand interaction: When ligand is in great excess, what equation can be used to determine the binding relationship without using protein measurements?

A

[PL]/[P]T = [L]T/[L]T + kd

257
Q

In a protein ligand interaction: Why is an excess of ligand more often used than an excess of protein?

A

Proteins are often more expensive than small molecules, furthermore it’s more difficult to measure protein concentration, rather than ligand concentration.

258
Q

In a protein ligand interaction: What assumptions are made for the equation
[PL]/[P]T = [L]T/[L]T + kd
to be valid?

A

When [L]»[P]
[L]T = [L] + [PL]
[PL] =~ 0, therefore,
[L]T =~ [L]

259
Q

What method can be used to produce a universal signal to monitor binding?

A

Titration calorimeters can measure the output of binding enthalpy, and-so

260
Q

In a bimolecular reversible reaction: When plotting the Kobs at different concentrations of [B] what is the equation for the produced straight line graph? (pseudo first order)

A

Kobs = K+1[B] + K-1
y = ax + b

261
Q

What is the importance of using both analytical and numerical methods of checking kinetics?

A

The comparison between values of the two allows for the evaluation of the analytical method used, providing information on whether or not the model used to analyse the reaction is accurate/appropriate.

262
Q

Why is it difficult to deduce the k-1 of a bimolecular reversible reaction when using a spectrophotometer?

A

The signal strength is dependent on the change in concentration (often of the ligand); with the total change decreasing as less binding continues and approaches 0. Therefore measurements become harder the closer you get to the y-axis, and thus the k-1 must be extrapolated from the data, whereas the K+1 is more accurately known.

263
Q

What model is used to describe simple enzyme reactions?

A

Michealis Menton.

264
Q

What is the advantage of the Michealis Menton in modelling of simple enzyme reactions?

A

The model provides a way to describe how the rate of enzyme catalysed reactions change with changes to concentrations.

265
Q

What is the disadvantage of the Michealis Menton in modelling of simple enzyme reactions?

A

The model is too simple, not acknowledging the EP complex, intermediates, and the dissociation of the product being irreversible rather than reversible.

266
Q

In a simple enzyme reaction: What is the equation to find the rate of product formation?

A

d[P]/dt = K+2[ES]

267
Q

In a simple enzyme reaction: What assumptions allow for [ES] to be found?

A

K+2 (which produces E+P) is assumed to be much less than K-1 (Where ES dissociates); this causes the reaction to be similar to Protein Ligand interactions, or the Bimolecular Reversible.

268
Q

In a simple enzyme reaction: What equation is used to find [ES]?(assuming K+2«K-1)

A

([E]eq x [S]eq)/[ES]eq = K-1/K+1 = Km.
Therefore [ES] = [E]T x [S]/([S] + Km)

269
Q

In what conditions can the michealis constant be used as a dissociation constant?

A

When K+2 &laquo_space;K-1 in a simple enzyme reaction.

270
Q

In a simple enzyme reaction: What is the rate equation?

A

v = K2[ES] = K2 x [E]T x [S]/([S] + Km) = (Vmax x [S])/ ([S] + Km)

271
Q

In a simple enzyme reaction: What is Kcat?

A

Kcat is the catalytic rate constant of an enzyme; In simple enzyme reactions where K+2&laquo_space;K-1, Kcat = K+2.

272
Q

In a simple enzyme reaction: What is the Vmax value?

A

When [S]»Km:
Kcat x [E]

When [S]«Km:
(Kcat/Km) x [E]T x [S]

273
Q

What is unique about the properties

A
274
Q

What equation is used when the assumption K+2«K-1 is not true?

A

Can perform the reaction under conditions wherein the rate of change of the enzyme/substrate complex is 0, by maintaining a constant excess of substrate.

275
Q

In a simple enzyme reaction: when K+2 &laquo_space;K-1 is not true, what is assumed about the rate of change of ES?

A

d[ES]/dt = 0

276
Q

In a simple enzyme reaction: when K+2 &laquo_space;K-1 is not true, what is the rate of formation of ES?

A

K+1 x [E] x [S]

277
Q

In a simple enzyme reaction: when K+2 &laquo_space;K-1 is not true, what is the rate of loss of ES?

A

K-1 x [ES] + K+2 x [ES]

278
Q

In a simple enzyme reaction: when K+2 &laquo_space;K-1 is not true, what is the equation linking the rate constants of ES formation and loss?

A

K+1 x [E] x [S] = (K-1 + K+2) x [ES]

or when rearranged,

[E][S]/[ES] = (K-1 + K+2)/K+1) = Km

279
Q

In a simple enzyme reaction: when K+2 &laquo_space;K-1 is not true, what is the Kcat value?

A

K+2

280
Q

In a simple enzyme reaction: when K+2 &laquo_space;K-1 is not true, what is the initial rate equation?

A

Vi = Kcat[E][S] / (Km + [S])

281
Q

In a simple enzyme reaction: when K+2 &laquo_space;K-1 is not true, what is the significance of the Kcat/Km term?

A

As K+2 becomes much greater and K-1 and K+1 -> the fraction will tend to the value 1.

282
Q

Why does initial rate not reflect the true time 0 rate?

A

Because the measurement will be taken milliseconds after the reaction.

283
Q

What is the pre-steady state period?

A

The time at the start of a simple enzyme reaction for the enzyme to become full occupied.

284
Q

What enzymes are used to reduce carbon chains in the FAS cycle?

A

To get rid of the intermediate formed with two keto groups, following the work of the synthetase, to synthesise a chain composed of only CH2 groups, the enzyme beta-ketoacyl reductase, beta-hydroxyacyl dehydratase (not a reductase), and enoyl reductase.

Year 2: Semester 2 (2 decks)

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