Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

Enzymes & Metabolism > Enzyme Mechanisms > Flashcards

Enzyme Mechanisms Flashcards

1
Q

Mechanism of Peptide Bond Hydrolysis

A
  • hydrolyse peptide bond into a carboxyl and amine group
    1. lone pair of nucleophile attacks sp2 hybridised carbonyl carbon to form bond
    2. loss of electrons causes positively charged attached nucleophile
    3. bond formation breaks octet rule around carbon so electrons move to oxygen
    4. formation of tetrahedral oxyanion intermediate
    5. electrons flow back onto carbon to reform double bond and leaving group gains electrons to leave (forms negative charge)
  • active site of enzyme contains serine residue, and the OH acts as a nucleophile
  • base used to deprotonate oxygen to promote nucleophilicity
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

Type sof Serine Proteases

A
  • Chymotrypsin
  • Trypsin
  • Elastase
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

Catalytic Triad

A

Active site contains triad of residues: serine, histidine, asparagine

  • Serine acts as a nucleophile
  • histidine acts as a base to pull proton off serine (must be activated to make it a better base due to its low pka)
  • deprotonated aspartate forms a charge-charge interaction via H bond with histidine stabilising it to make it a better base
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

Catalytic Mechanism of Active Site

A
  1. nucleophilic attack by Serine to form tetrahedral intermediate (histidine is a general base and asparagine exerts electrostatic effect)
    - OH electrons attack carbon center and electrons move onto oxygen to give oxyanion
  2. decomposition of tetrahedral intermediate to give acyl-enzyme intermediate. protonated histidine is a general acid to form amine leaving group
  3. histidine acts as a general base to promote nucleophilic attack on water on the acyl-enzyme to form second tetrahedral intermediate
    - histidine protonated and nucleophilicity of water increased: nucleophilic attack on carbonyl to form another tetrahedral intermediate and oxyanion
  4. decomposition of intermediate to give resting enzyme and carboxylic acid. protonated histidine is a general acid
    - serine side chain picks up proton from His to be a better leaving group and release carboxylic acid as second product
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

Oxyanion Hole

A

Stabilises Transition State

  • exploits the change in hybridisation of carbon from sp2 to sp3 tetrahedral intermediate
  • negative oxygen in oxyanion intermediate moves into hole and H bonds to NH backbone groups (glycine and serine), reducing free energy of activation
  • in MM complex, trigonal carbon of scissile peptide bond is conformationally constructed from binding into the oxyanion hole
  • additional H bond to glycine also made
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

Substrate Specificity of Serine Proteases

A

Enzymes have a different specificity for the amino acid preceding the scissile bond (P1 position complements S1 specificity pocket)
Chymotrypsin: bulky residues
Trypsin: positively charged residues
Elastase: small neutral residues

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

Evolution of the Catalytic Triad

A
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

Similarity to other Proteases

A

eg. cysteine, aspartyl, metalloproteases
- all nucleophilically attack carbonyl
- all protonate the amine leaving group

Commonalities

  1. presence of nucleophile for attack
  2. presence of charges to polarise carbonyl group and stabilise tetrahedral intermediate
  3. presence of proton donor to make NH better leaving group
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

Zymogens

A
  • serine proteases are synthesized as larger precursors molecules activated by proteolytic cleavage
  • specificity pockets and oxyanion holes improperly formed
  • low levels of catalytic activity resulting from inability to bind substrate productively/stabilise TS
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

Trypsinogen

A
  • activated by cleavage after Lysine
  • trypsin is autocatalytic and catalyses its own activation-
    pancreatic trypsin inhibitor prevents inappropriate activation
  • trypsin also activates chymotrypsinogen
  • cleaves chymotrypsinogen to pi-chymotrypsin
  • pi-chymotrypsin is auto catalytic and cleaves itself to active a-chymotrypsin held together by disulfide bridges
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

Chemical Labelling Studies

A
  • diagnostic test for presence of active serine of serine proteases is its reaction with DIPF
  • reacts with fluoride to form DIP-enzyme and hydrogen fluoride irreversibly
  • ie. serine side chain nuclephilically attacks P center to eject fluoride
  • this irreversibly inactives the enzyme
  • DIPF reacts only with serine 195, proving it is a key residue, ie. part of the catalytic triad
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

Affinity Labelling

A
  • His 57 was identified by affinity labelling using a substrate analogue (TPCK)
  • TPCK has saturated carbon susceptible to SN2 mechanism attack
  • histidine is basic and reacts with carbon
  • C1 ejected as leaving group
  • TPCK resembles chymotrypsin substrate but is chemically modified to react irreversibly when bound in active site (analog forms stable covalent bond with susceptible group)
  • showed that histidine is a key residue
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

Initial Burst Experiment **

A
  • Conducted an assay using absorbance spectroscopy and found there were two phases to product activity
    Burst Phase: initial phase where product is formed in equal amounts to enzyme on mole basis
    Steady State Phase: product is produced at constant rate dependent on enzyme concentration
  • ie. there is an initial burst of product formation

Interpretation of this experiment:

  • fast initial burst of product to generate covalently attached acyl enzyme intermediate
  • slower regeneration of enzyme is RDA of catalytic cycle//limits catalysis in subsequent turnovers
  • ‘ping pong’ mechanism???
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

Lysozymes

A
  • example of strain
  • hydrolyze B (1-4 carbon) glycosidic linkage eg. in peptidoglycan chains
  • cleaves linkage between positions D and E
  • N acetylmuramic acid to N acetylglucosamine
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

HEW Lysozyme

A
  • single polypeptide chain cross linked with 4 disulfide bonds
  • 5 helical segments and 3 antiparallel B sheets
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

Structure of Lysozyme

A
  • cleft (substrate binding site) traversing one face
  • binds (NAG) 6 carbon sugar
  • favorable binding of other sugars drives residue D binding (positive delta G)
  • fourth residue distorts into a half chair conformation as it has steric hindrance in position D between C6/O6, and glutamine, trypsinogen, acetamido groups
  • distortion moves O5 down and C5 up and moves C6 group into an axial position to H bond with backbone residues
  • distortion stabilised by H bonding between D ring O6 and backbone NH of valine
17
Q

Catalytic Residues of Lysozyme

A
  • Asparagine surrounded by polar residues (H bond network) is unprotonated and negatively charged
  • Glutamine is in a non polar pocket meaning it remains protonated at lower pH
18
Q

Non-Catalysed Lysozyme Mechanism

A
  • hydrolysis of acetal to hemiacetal
  • protonation of oxygen to make better leaving group
  • carbocation favored due to + charge delocalisation and resonance
  • drives departure of neighboring sugar
  • Sn1 type mechanism
19
Q

Phillips Mechanism (SN1 like)

A
  1. glutamine (protonated) acts as a general acid and protonates bridge oxygen of glycosidic bond
    - oxygen’s lone pair attacks C1
  2. glycosidic bond cleaves leaving a positively charged D ring oxonium ion stabilised by favorable electrostatic interactions with negatively charged Asparagine carboxylate and enzyme induced distortion of the D ring to enhance stabilisation
  3. depronotated glutamine is a general base and activates water to perform addition reaction at carbocation
  • D ring oxonium intermediate stabilised by resonance in half chair but not chair conformation, lowering delta G
  • p orbitals lie at the same level to allow good overlap of generate a pi bond
  • only occurs in half chair conformation as in chair conformation the oxygen is too high
20
Q

Koshland Mechanism (SN2 like)

A
  1. first SN2 like step, negatively charged aspartate carboxylate acts as a nucleophile to displace first product
  2. in concerted mechanism, glutamine is a general acid and protonates bridge oxygen of glycosidic bond
    - glutamine has high pka to keep it protonated
  3. glycosyl-enzyme covalent intermediate (asparagine residue) is formed - this is covalent catalysis
  4. second SN2 like step, deprotonated glutamine is a general base to activate water to perform nucleophilic SN2 reaction to displace asparagine to release second product
21
Q

Retention of Conformation (phillips mechanism)

A

OH occupies same place as oxygen linkage between 2 sugars
- aspartate residue has electrostatic stability role and sterically hinders active site so hydroxide can only attack from the side

22
Q

Hexokinase

A
  • example of induced fit

- phosphorylation of glucose to form Glc-6-P in glycolysis

23
Q

Mechanism of Hexokinase

A
  1. nucleophilic attack of C6-OH group on y phosphate of an Mg2+ - ATP complex
    - this is metal ion catalysis
    - base needed to deprotonate OH group to increase nucleophilicity - allows more negative charge to develop
  2. the negative charges of ATP hinder nuclephilic attack on phosphorous center
    - Mg ion neutralises negative charges
  3. P double bond is a polarised system with electronegative oxygen, meaning the P is partially positive, electrophilic, and susceptible to nucleophilic attack
  4. SN2 mechanism: attack by oxygen leads to bond formation and ADP leaving group as electrons flow back onto the ester linkage
24
Q

Alternative Hexokinase Mechanism

A
  1. addition elimination reaction
  2. bond formation between oxygen and phosphorus
  3. two electrons move onto oxygen to form oxyanion
  4. addition reaction then oxyanion decomposition to eject O- of ADP
25
Q

Side Reaction of Hexokinase

A
  • competing reaction of ATP reacting with OH of water

- nucleophilic attack by water to release inorganic phosphate

26
Q

Induced Fit of Hexokinase

A
  • prevents interference of water
  • 2 flexible lobes of enzyme
  • movement of lobes upon substrate bonding squeezes out water preventing ATP hydrolysis in side reaction
27
Q

Lactate Dehydrogenase

A
  • stereopsecific formation of lactate from pyruvate
  • hydride attacks carbonyl group of pyruvate to generate lactate
  • reduction of NAD to NADH
  • only forms one stereoisomer of lactate
28
Q

Hydride reduction of carbonyl groups

A
  • hydride attack on carbonyl carbon
  • electrons of double bond attack acid to protonate oxygen
  • reduction reaction gives secondary alcohol
29
Q

Hydride Attack and Chirality

A

Hydride Ion can attack from the Re (top) or Si (bottom) face of an sp2 trigonal planar molecule

30
Q

Pro-R and Pro-S

A

Describe 2 identical substitutions to a sp3 hybridized atom

31
Q

Cahn Ingold Prelog Priority Rules

A
  1. atoms attached to chiral center ranked in terms of atomic number with highest given priority
  2. if there is a tie, atoms bonded to tied atoms are considered
  3. ghost atoms used for multiple bonds
  4. molecules viewed with lowest priority group pointing away

R stereoisomer: groups 1,2,3 joined by clockwise rotation (RE face)
S stereoisomer: groups 1,2,3 joined via anticlockwise rotation (SI face)

32
Q

Dehydrogenase Coenzymes

A
  • NAD and NADH used as coenzymes
  • oxidised form: aromatic
  • reduced form: not aromatic
  • nictotinamide ring carries hydride anion
  • C4 in reduced form is prochiral center - replacement of one hydrogen would cause chirality
33
Q

Hydride Formation from Reduced form of NAD

A
  1. lone pair on nitrogen move around ring system
  2. breaks octet rule around C4, so hydrogen leaves
  3. hydride leaving group nucleophilically attacks carbonyl of substrate
    - concerted mechanism
34
Q

Reaction Mechanism of Lactate Dehydrogenase

A

only pro-R hydrogen is transferred and always to the RE face of the pyruvate (absolute stereospecificity)

  1. enzyme pins pyruvate into the active site so only one face of the molecule is accessible for attack
  2. carboxylate group has charge charge interactions with protonated arginine side chain
  3. H bonds between positive arginine side chain and histidine side chain with carbonyl group
    - pyruvate fits into a specific geometry within active site (carboxylate bond specifically)
  4. hydride attacks RE face
  5. histidine is a general acid to protonate oxyanion species (electrons pushed onto oxygen as hydride attacks due to octet rule)
  6. pro R hydrogen transferred because it is closer to carbonyl carbon
    - stereospecific transfer of pro R hydrogen
  7. free rotation about R group
    - interaction between primary amide and active site residues pinning ring and fixing it into place so that only pro R hydrogen is able to attack
35
Q

Rossman Fold

A
  • conserved nucleotide binding domain found also in other dehydrogenases
36
Q

ProChiral Differentation

A
  • MM complex has geometric and electronic specific that confers substrate specificity
  • differentiation between seemingly chemically identical groups

Enzymes & Metabolism (12 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions