Enzymes

Question 1

Enzyme-substrate complementarity

Answer 1

Substrate and residues in active site as close together as possible for maximum binding energy (snug fit)
Greater complementarity + greater binding energy

Question 2

Factors involved in binding substrate

Answer 2

H bonds
Electrostatic
Hydrophobic
VDW

Question 3

What is Kcat

Answer 3

Turnover number

Question 4

What is Kcat/Km

Answer 4

Indicated whether an enzyme-catalysed reaction is diffusion controlled
I.e. chemical+release steps are so fast that enzyme is just waiting to encounter substrate
10^8 to 10^9 M-1S-1

Question 5

Transition state theory

Answer 5

Transition state represents the most unstable species in a reaction pathway where chemical bonds are being broken and made and is located at the peak of the reaction profile
Intermediates have fully formed bonds and occupy troughs in the profile
Kcat/Kuncat = e^change in binding energy/RT
Active site of an enzyme has evolved to bind TS more tightly than the substrate
Gives maximum binding energy

Question 6

General acid catalysis

Answer 6

Process whose rate is dependent on the concentration of all acids present in the reaction not just {protons] of aqueous reactions, e.g. a side chains

Question 7

General base catalysis

Answer 7

Process whose rate is dependent on the concentration of all bases present in the reaction, not just [OH-] if aqueous reaction

Question 8

Specific acid / base catalysis

Answer 8

Reactions whose reactions are just dependent on [H+] or [OH-]

Question 9

Concerted general acid-base catalysed reaction

Answer 9

Both processes occur simultaneously, only one TS, proton delivered and accepted at same time

Question 10

PH dependency of acid-base catalysed reactions

Answer 10

Reversible reaction where aa side chains are deprotonated
Above pKa= deprotonated
Must be in correct protonation state for enzyme to be active

Question 11

Effect of environment on pKa values

Answer 11

PKa values can be shifted by micro environment
Polarity + presence of charges
Hydrophobic region = uncharged species favoured = increase pKa
+ve charge nearby = -ve charge stabilised = decreased pKa
-ve charge nearby = -ve charge destabilised = increased PKa

Question 12

Covalent catalysis

Answer 12

Formation of covalently bound enzyme intermediate
Form from reaction of the substrate with an aa side chain or a coenzyme
Aa side chains can act as nucleophiles but only reactive when deprotonated
Important in group transfer reactions - 2 substrate reactions that usually involve transfer of one group from one substrate to another
Important in making substrate more reactive
E.g. formation of schifi bases to activate carbonyl groups

Question 13

chemical trapping of enzyme intermediates

Answer 13

React enzyme with NaBH4, source of hydride ions
Forms amine, non-reactive , prevents further catalysis
Enzyme inactivated as modified form of substrate is permanently attached to Lys side chain

Question 14

Electrostatic catalysis

Answer 14

Presence of charges and oriented dipoles within the active site can stabilise TS by solvation
TS accumulates charge, surround TS with opposite charge to get favourable charge-charge interactions to get binding energy
Coulombic interactions are more effective in non-polar environments as they have lower dielectric constant

Question 15

Metal ion catalysis

Answer 15

Metalloenzymes containing tightly bound ions e.g Fe2+ or Mg2+
Metal-activated enzymes loosely bind ions from solution e.g. Na+ , K+

Question 16

What are metal ions involved in

Answer 16

Electrostatic stabilisation and charge screening
Highly effective because of high charge that is pH independent
E.g. Mg2+-ATP complex in kinases
Redox reactions
Binding and orientation of substrates
Act as Lewis acids and can form dative or covalent coordinate bonds
Promote nucleophilic catalysis by activating water bound molecules

Question 17

Catalysis through orientation and proximity effects

Answer 17

Rate enhancement obtained by taking 2 reactants out of solution and placing them next to each other in the enzyme, raising the local concentration of each reactant
Binds substrate in precise geometry so orbitals have correct orientation

Question 18

Entropy changes in bimolecular reactions

Answer 18

A + B —> A-B has large -ve delta s
A + B —> AB in enzyme —> A-B smaller -ve S so more favourable
Enzyme can tether reactants together to increase local conc

Question 19

Serine protease catalytic triad

Answer 19

Asp-102
His-57
Ser-195

Question 20

Serine protease mechanism

Answer 20

1)Nucleophilic attack of serine to form first tetrahedral intermediate
His-57 general base
Asp-102 electrostatic effect
2) decomposition of first tetrahedral intermediate to give Acyl-enzyme intermediate
His-57 general acid to form amine leaving group
3) His-57 acting as general base promotes nucleophilic attack by water on the Acyl-enzyme to form second tetrahedral intermediate
4) decomposition of intermediate to give resting enzyme and carboxylic acid
Protonated his-57 acts as a general acid

Question 21

Serine protease oxyanion hole

Answer 21

Acts to stabilise transition state
In michaelis complex, trigonal carbon of scissile peptide bond is conformationally constrained from binding into oxyanion hole
In tetrahedral intermediate, the oxyanion species enters the hole and makes favourable H bonds to the backbone NH groups of Gly193 and ser 195
An additional H bond to gly193 is also made
Favourable binding interactions reduce energy of activation
Carbonyl oxygen shifts into cavity
Charged group stabilised by residues in enzyme
Only occurs in TS

Question 22

Specificity of serine proteases

Answer 22

Determined by nature of S1 pocket and p1 residues of substrate
Chymotrypsin cleaves bulky P1, Phe, trp, Tyr
Trypsin has asp in pocket so cleaves +ve P1, Arg, Lys
Elastase has 2 valines in pocket s cleaves small, neutral P1, Ala, gly, ser, val

Question 23

Divergent vs. Convergent evolution

Answer 23

Divergent is after a gene duplication event
Only need one copy of the gene so now second can mutate without causing harm
Produced trypsin, chymotrypsin, elastase
Convergent evolution produces same triad but with completely separate route

Question 24

Kinetics of chymotrypsin

Answer 24

Shown with pre-steady state kinetics
Fast initial burst of product to generate covalently attached Acyl-enzyme intermediate
Slower regeneration of enzyme is RDS of catalytic cycle and limits catalysis in subsequent turnovers

Question 25

Cysteine proteases

Answer 25

Similar to serine Use thiol group which has lower pKa than ser No aspartate as it doesnt need a base to pull off the proton

Question 26

Aspartyl proteases

Answer 26

Water attacks directly Aspartate in conjugate base form to pull off a proton Second asp in protonated form ti H bond with oxyanion species to stabilise it 2 asp residues must have different pKa values

Question 27

Metallo proteases

Answer 27

Similar to carbonic anhydrase Tightly bound metal ion can bind water Appropriately positions H2O to attack Base close by to pull off proton Electrostatic interaction between Zn2+ and oxyanion

Question 28

Hydrolysis of peptide bond is favoured by

Answer 28

Presence of nucleophile to attack carbonyl Presence of charges to polarise carbonyl and stabilise tetrahedral intermediates Presence of proton donor to make -NH a better leaving group

Question 29

How is trypsinogen activated

Answer 29

Serine proteases are synthesised as larger precursor molecules called zymogens in pancreas that show little activity Trypsinogen activated by cleavage after lys15 Initially cleavage is catalysed by a serine protease under hormonal control Trypsin can then catalyse it’s own activation (autocatalytic) Inappropriate activation in the pancreas is prevented by synthesis of pancreatic trypsin inhibitor which binds tightly to trypsin Proelastase is activated in a similar manner

Question 30

Activation of chymotrypsinogen

Answer 30

Trypsin activates chymotrypsinogen by removing dipeptides Ser14 Arg15 and Thr147 Asn148 The protein is linked by disulphide bridges

Question 31

What does lysozyme do

Answer 31

Destroyed bacterial cell walls by hydrolysing the beta 1-4 glycosidic linkage between NAM and NAG sugar units

Question 32

Groove of lysozyme

Answer 32

6 sugar units a-f fit into groove of enzyme Cleavage between e and d All 6 units needed to drive catalysis Binding energies of all units apart from d are positive D has a high, unfavourable binding every Explains why you need more sugar as you need favourable binding to drive unfavourable binding Binding to sub site d involves structural distortion (strain) of the sugar Sugar in subiste d adopts half chair conformation

Question 33

Philips mechanism for lysozyme

Answer 33

SN1-like 1) glu35 acts as a general acid and protonates bridge oxygen of the glycosidic bond Unusually high pKa to ensure in protonated state 2)glycosidic bond cleaves, leaving a positively charges D ring Oxonium ion which is stabilised by favourable electrostatic interaction with -ve asp52 and the enzyme induced distortion of the D ring to enhance resonance stabilisation 3) glu35 in deprotonated state acts as a general base to activate water to perform addition reaction at carbocation

Question 34

Lysozyme koshland mechanism

Answer 34

2 back to back sn2-like steps 1) -ve asp52 acts as a nucleophile to displace the first product In a concerted mechanism, glu35 acts as a general acid and protonated bridge oxygen of the glycosidic bond Glu35 has an unusually high PKa to ensure in protonated state and reaction aided by distortion of the d ring 2) Glycosyl-enzyme intermediate is formed 3) glu35 in deprotonated state acts as a general base to activate water to perform nucleophilic sn2 reaction to displace asp52 to release the second product *detection of the Glycosyl intermediate suggests this mechanism operates

Question 35

Hexokinase

Answer 35

First step of glycolysis Sn2 reaction Phosphoryl group is transferred into oxygen Competing reaction is H2O hydrolysing atp

Question 36

Mechanism of Hexokinase

Answer 36

Penetrate -ve charge barrier on p by forming dative covalent bonds with mg2+ to give neutral species Nucleophilic attack of the c6-oh group of glucose onto the gamma phosphate of an mg2+-atp complex (metal ion catalysis) Hexokinase is an example of induced fit Movement of lobes upon substrate binding squeezes out water, preventing the possible hydrolysis of atp in competing side reaction Glucose partially buried

Question 37

Lactate dehydrogenase role

Answer 37

Catalyses stereospecific formation of lactate from pyruvate In principle reaction could form 2 enantiomers Only L isomer is formed In the hydride reduction of a carbonyl, hydride can attack from above or below the plane

Question 38

R and S

Answer 38

R stereoisomer is 1,2,3, clockwise S stereoisomer is 1,2,3, anticlockwise Same logic for re and si

Question 39

Pro r and pro s of NADH

Answer 39

C4 is prochiral

Question 40

Mechanism of lactate dehydrogenase

Answer 40

Reactants are pinned in precise geometry in the enzyme by Arg resides forming hydrogen bonds Only the pro-r hydrogen is transferred and always to the re face of pyruvate An example of absolute Stereospecificity Geometric and electronic complementarity in the michaelis complex confers substrate specificity

Question 41

Allosteric activator

Answer 41

Stabilises relaxed form

Question 42

Allosteric inhibitor

Answer 42

Stabilises tense form

Question 43

ATCase role

Answer 43

Catalyses committed step in pyrimidine biosynthesis Carbamoyl phosphate + aspartate —> N-carbamoyl Aspartate

Question 44

ATCase structure

Answer 44

3 regulatory dimers 2 catalytic trimers Zinc motif binds subunits Active site contains residues from more than one subunit Side chains contribute to specific binding Each binding site is a combination of 2 subunits

Question 45

Control of ATCase

Answer 45

Pala is competitive inhibitor Also controlled by feedback inhibition As [CTP] increases, rate of N-carbamoyl aspartate formation decreases R state favoured by substrate binding T state favoured by CTP binding CTP is negative regulator ATP is positive regulator

Question 46

Sigmoidal kinetics of ATCase

Answer 46

Active sites cooperate Titration behaviour when substrate is added Cooperativity- binding of ligand at one active site affects binding at other sites Model for cooperativity is 2 michaelis menten enzymes with different km values

Question 47

Key MWC parameters

Answer 47

L = [T0]/[R0] Larger L means t to R equilibrium lies further towards t state C = kR/kT Smaller value of c means greater affinity of the substrate for R state compared to T state. Higher c means smaller difference in binding between t and r forms so less of a cooperative transition N Higher value n = more substrate can bind = T-R equilibrium can be shifted more towards R state. Smaller n means less cooperative effect

Question 48

Structure of Haemoglobin and myoglobin

Answer 48

Mb is monomeric and stores o2 in tissues Hb is an alpha2beta2 tetramer and carries o2 in blood Mb has single O2 binding site

Question 49

Ligation oh haem

Answer 49

Haem has 6 potential ligand positions 4 are occupied by planar porphyrin ring nitrogen’s 1 ligated by proximal histidine In absence of o2, Fe is below the plane of the ring When o2 binds, d orbitals contribute to bonds so VDW radius shrinks and Fe fits into plane of ring Protein responds to motion as proximal histidine is pulled up by 0.4A

Question 50

Resonance structures of bound O2

Answer 50

Fe2+—O=O dioxygen Fe3+—O—O- superoxide (deadly for cells) ferrous(Fe2+) is stabilised in Mb by distal histidine Structure of Haemoglobin prevents oxidation of Fe

Question 51

Quantification of ligand binding to single independent site

Answer 51

KD = [P][L]/[PL] [PT]=[P]+[PL] KD = [L] that gives 50% binding

Question 52

Cooperativity of Hb binding

Answer 52

Sigmoidal curve Hb has 4 binding sites 2 pairs of alpha beta dimers Alpha1beta1 and alpha2beta2 Strong interactions between alpha1beta1 and alpha2beta2 interfaces Strong interactions between alpha and beta Alpha helices lean against each other and push and pull

Question 53

Hill coefficient

Answer 53

Measure of cooperativity If n>1 it suggests 2 or more binding sites and +ve cooperativity <1 is negative cooperativity 1= no cooperativity At extreme values, Hb is mostly T state, small changes in O2 pressure wont produce much R When almost saturated any changes in occupation wont produce more R as hardly any T left

Question 54

What causes T to R transition in Haemoglobin

Answer 54

The fe2+ atom moves into the plane of the haem upon oxygen binding Helix containing his ligand moves Movement of the helix alters the interface between the alpha beta pairs Alpha beta pairs slide and rotate upon formation of the R state

Question 55

Allosteric regulation of Hb

Answer 55

Negative regulators promote O2 release 2,3-bisphosphoglycerate (produced in RBC glycolysis) CO2 H+

Question 56

How does 2,3-BPG promote oxygen release from Hb

Answer 56

Stabilises T form BPG occupies central hole in t form Increases BPG levels allow acclimation to high altitude More bpg shifts binding curve to the right, less Hb occupied Foetal Haemoglobin has alpha2gamma2 form with reduced affinity for BPG His 143 mutated to serine Still good binding interactions Binding curve shifts left as r state stabilised so O2 flows into blood circulation foetus

Question 57

PH and CO2 in oxygen release

Answer 57

Decreasing pH (Bohr effect) and increasing co2 levels promotes O2 release Co2 is a weak acid so creates protons, causing binding curve to shift right as t state stabilised Protonation of his146 by lowering pH promotes stabilisation of T form of deoxyhaemoglobin

Question 58

Transport of CO2 out of body

Answer 58

CO2 reacts with N terminal amino groups to generate carbamate Carbamate transports CO2 out of body attached to Hb Most CO2 transported through body dissolved in blood Most transported to lungs in form of bicarbonate produced in RBS and then released into blood plasma

Question 59

What is a cofactor

Answer 59

Organic molecules/ions required by an enzyme for activity

Question 60

What is holoenzyme and apoenzyme

Answer 60

Holoenzyme is cofactor + protein Apoenzyme is protein alone (inactive)

Question 61

What is a vitamin

Answer 61

Small organic molecules that are essential for growth but unable to be synthesised by the organism so must be present in the diet

Question 62

What is biotin

Answer 62

Has an imidazoline ring that is cis-fused to a tetrahydrothiophene ring with a valerete side chain Valerete side chain holds biotin into active site as prosthetic group Covalently attached to enzyme In the carboxybiotinyl enzyme, N1 of the biotin ureido group is in the Carboxylation site Valerete side chain has amide link to lysine side chain in enzyme Biotin binds co2 and delivers it to relevant active site

Question 63

Role of biotin in pyruvate carboxylase

Answer 63

Biotin moves co2 huge distances Pyruvate carboxylase has 2 subunits 1 domain for Carboxylation 2nd domain for carboxyltransferase 3rd domain with biotin attached on swinging arm Swinging arm swings between 2 subunits Connects 1st domain of one subunit with second domain of the other

Question 64

What is pyridoxal phosphate (PLP)

Answer 64

Has reactive aldehyde group and acidic phenolic group 6 member ring with a nitrogen N lone pair not delocalised in ring so n can act as a base Reactive aldehyde reacts with amino groups to form schiff base Acidic phenol has lower pKa so can be deprotonated Can H bond to intermediates or play role in acid-base catalysis Forms tautomer when H from phenol moves onto N NH+ can then act as temporary e- sink to stabilise carbanions Stable tautomeric form allows PLP to act as an e- sink during reactions

Question 65

Aminotransferase reaction

Answer 65

Aa1 + enzyme-PLP —> alpha keto acid 1 + enzyme-PMP Amine is transferred from aa1 to pyridoxal group Transfer of amino acid is covalent catalysis PMP shows it has attached amino group Alpha keto acid 2 + enzyme-PMP —> aa2 + enzyme-PLP Amine transferred from pyridoxamine group to alpha keto acid 2 Group transfer reaction is via ping pong mechanism

Question 66

How is amino group transferred in aminotransferase reaction

Answer 66

Transferred by tautomerisation of schiff base Proton needs to be weakly acidic Pyridine ring stabilises carbanion Proton is detached and moved to another carbon because of octet rules the double bond has to shift

Question 67

Mechanism of aminotransferase

Answer 67

Transamination Tautomerisation Hydrolysis

Question 68

How is aminotransferase intermediate stabilised

Answer 68

Planarity is important for resonance stabilisation Essential for pi orbital overlap Configuration of the intermediate is tightly controlled by the architecture of the enzyme active site Carboxylate of alpha carbon is held in place by coordination to an Arg residue H bond keeps system planar The proton that is being transferred can only come from one direction so only one stereoisomer produced All aas have 2S configuration apart from Gly and Cys Elimination occurs perpendicular to the plane of the delocalised system Rotation around C-N bond brings different groups perpendicular to delocalised pi system When bond is broken C forms p orbital that can delocalise into system