Brainscape's Knowledge GenomeTM


Top Flashcards (Certified)
All Flashcards

Y2 Biochemistry > Enzyme Mechanisms > Flashcards

Enzyme Mechanisms Flashcards

1
Q

What are the three things reaction mechanisms determine?

A

Where electrons are
Which bonds are broken/made
Flow of electrons

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

Describe BrØnsted acids and bases

A

Acid- proton donor
Base- proton acceptor

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

Describe lewis acids and bases

A

Acid- can form coordinate bonds with lone pairs
Base- has lone pairs available for sharing

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

What is the pH equation with a certain pKa?

A

pH = pKa + log10([A-]/[HA])

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

What factors determine acidity of an organic compound?

A

Strength of Y-H bond
Electronegativity of Y
Factors to stabilise Y- compared to YH
Nature of the solvent

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

What does pKa depend on?

A

Temperature
Ionic strength of solvent
Microenvironment

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

Describe the protonated and deprotonated forms of histidine

A

The protonated imidazolium cation is stabilised by 2 resonance structures
Deprotonated imidazole has 2 tautomers where either N is protonated

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

Why is cysteine more acidic than serine?

A

The S valance electrons are further from the nucleus than in O, so electrons are easier to donate

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

Describe RNase structure

A

V shaped
3-stranded antiparallel β-sheets
N-terminal helix with His12
4 SS bridges

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

What do RNases cut?

A

They act as endonucleases on ssRNA
Cleave P-O5’ bond
Cuts after a pyrimidine base (U or C), suggesting there must be a recognition site

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

Describe the key catalytic intermediate of the RNase reaction

A

2’-3’ cyclic nucleotide. This is cleaved in the second step of the RNase reaction

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

How were kinetic studies used to predict the active site of RNase?

A

The pH profile of Vmax is around 6-7, suggesting there might be 2 His residues in the active site- one acid, one base

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

How were His12 and His119 identified as active site residues in RNase?

A

Modified RNase could only have one key His modified at a time, but not together, His119 was 88% and His12 12% of the time, so one acted as an acid, the other a base

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

Describe the RNase A active site residues

A

His12 and His119 are directly involved in cleavage
Lys41 stabilises negatively charged phosphates in the intermediate

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

Describe the RNase A specificity pocket residues

A

Phe120- van der waals contacts with the RNA base
Ser123 and Thr45 are involved in hydrogen bonding
The pocket is too small for purines (AG)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

What is the acidic active site residue of RNase?

A

His12

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

Give an example of a serine protease

A

Trypsin
Chymotrypsin
Elastase
Thrombin
Kallikrein
Compliment System
Blood clotting factors

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

What sort of reaction do serine proteases catalyse?

A

Hydrolysis of a scissile bond with water

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

How do the S1 and S1’ pockets bind to P1 and P1’ respectively?

A

S1 binds upstream and S1’ downstream of the scissile bond

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

How is chymotrypsin initially synthesized?

A

Inactive enzyme precursor (zymogen) called chymotrypsinogen

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

Where about in a polypeptide does chymotrypsin cleave?

A

After large aromatic residues (Phe, Tyr, Trp)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
22
Q

What is the catalytic triad in the chymotrypsin active site?

A

Ser195
His57
Asp102

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
23
Q

How can Ser195 be modified in chymotrypsin?

A

PMSF blocks serine proteases by modifying Ser195, inhibiting the enzyme

DIPF is a nerve gas which blocks serine proteases and related molecules

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
24
Q

How can His57 in Chymotrypsin be identified?

A

TPCK is a substrate analogue which binds at the active site and is nucleophilic attacked by His

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
25
Describe trypsin-like serine protease structure
Two β-barrels formed from 6 anti-parallel β-strands His57, Ser195, Asp102 catalytic triad Oxyanion hole Specificity pocket Main chain substrate binding
26
Describe how the chymotrypsin catalytic triad is stabilised
-ve Asp102 stabilises formation of +ve His57, so the proton from Ser195 can donate to His57 This makes Ser195 nucleophilic
27
Describe the chymotypsin oxyanion hole
Located near the carbonyl of the substrate scissile bond Backbone amide hydrogens of Ser195 and Gly193 point to the active site to stabilize the tetrahedral enzyme-substrate intermediate
28
Describe the chymotrypsin mechanism
1. His57 attacks Ser195 which attacks the carbonyl group of the substrate, forming a covalent bond. This forms a -ve tetrahedral intermediate stabilised by the oxyanion hole 2. Tetrahedral intermediate decomposes to the acyl-enzyme intermediate with His57 donating the proton 3. The enzyme is deacylated through another tetrahedral intermediate. Water is the nucleophile, Ser195 is the leaving group
29
What % identity do chymotrypsin and trypsin have?
40%
30
What residues are found in the chymotrypsin, trypsin and elastase specificity pockets?
Chymotrypsin: G266, S189, G216 Trypsin: G266, D189, G216 Elastase: V226, T216
31
How do serine proteases orient polypeptides for cleavage?
Bind to the side chains in the specificity pocket and recognise the main chain conformation.
32
How are serine proteases controlled?
Produced as zymogens (proenzymes) which require proteolytic cleavage of their precursors to activate
33
What is the protease activation cascade master activator?
Eteropetidase
34
how is α-chymotrypsin activated?
1. Chymotrypsinogen is partially activated by cleavage by trypsin to π-chymotrypsin. 2. This activates other π-chymotrypsin molecules to form α-chymotrypsin. 3. The A,B and C chain are connected by disulfide bonds
35
How is the chymotrypsin active site activated?
The N-terminal Ile16 on π-chymotrypsin ionically pairs to the Asp194 side chain. This changes the position of the Ser195 side chain for the correct geometry. The amides of residues 193 and 195 are oriented to form the oxyanion hole
36
Where does thrombin cleave fibrinogen?
Arg-Gly This forms plaques of insoluble fibrin, forming clots
37
How are serine proteases used in the blood clotting cascade?
Thrombin cleaves fibrinogen to form insoluble fibrin Positive and negative feedback form the activation and deactivation systems Plasmin/plasminogen system dissolves clots
38
Give an example of convergent evolution of serine proteases
Subtilisin is a bacterial protease with an unrelated sequence to chymotrypsin. However it has the Asp, His, Ser catalytic triad
39
Describe proteasomes
Intracellular complexes with proteolytically active sites in its interior cave belonging to N-terminal threonine hydrolases. 4 stacked rings of 7-fold symmetry
40
What sort of activity do the protease active sites of the inner (β) subunits of the proteasome have?
β1- caspase β2- tryptic β5- chymotryptic
41
How does bortezomic inhibit apoptosis?
Induces the ubiquitin-proteasome pathway by binding to the β5 subunit. It has a trigonal conformation which mimics a peptide bond. When boron is attacked by a nucleophile, it is converted from trigonal sp2 to tetrahedral sp3.
42
What cellular processes are associated with cysteine proteases?
Apoptosis, parasitic infections, virus maturation
43
Outline the action of a cysteine protease
1. Nucleophilic attack by a thiolate on C=O 2. Tetrahedral intermediate forms in the oxyanion hole 3. A water molecule is activated by His and attacks the carbonyl group 4. Second tetrahedral intermediate forms
44
What is Burkoldria?
B. pseudomallei. Potential bioterror weapon. Multi-drug resistant bacterium with no vaccine. Infection mimics TB 7 morphotypes with different patterns of gene expression
45
How does BPSL1549 from burkholdria compare to the CNF1 domain?
They have structural, but not sequence similarity. This is an example of convergent evolution. The catalytic His-Cys pair is conserved
46
Describe CNF1
114kDa pathogenic toxin expressed by some E.coli Deamidates key glutamine residues in small GTPases (Rho, Rac and Cdc42) Inhibits GTP hydrolysis, by deactivating GTPases. This leads to cytoskeleton remodelling
47
Describe BPSL1549
23kDa protein in Burkholdria. Unknown function and no sequence similarity to proteins outside of burkholderia. Deamidates Gln339 in RNA helicase eIF4A, preventing initiation-dependant protein translation.
48
What metal ion facilitates catalysis of the enolase superfamily?
Mg2+
49
Outline the mandelate racemase mechanism
Lys166 acts as a base to remove H from the chiral carbon. The OH then attachs the carbonyl C=O His297 acts as an acid and returns the proton
50
Describe the mandelate racemase active site
The Mg ion is coordinated by carboxylic acid groups on Asp/Glu Mg2+ stabilises the acid-carboxylate The L-proton faces Lys166 and catalytic histidine returns the D-proton on the opposite side
51
What is a common H- source in lab settings
BH4- (Borohydride)
52
Why are biological reductants better than BH4-?
They use cofactors which are directed by enzymes to target molecules, making them very specific
53
Describe the main features of an NAD ring
Net positive charge all over Phosphate backbone in nucleotides gives an overall negative charge
54
Why is hydride transfer very stereospecific?
The enzyme differentiates between hydrogens pro-R/pro-S on the carbon. Hydride transfers to a particular face of the nicotinamide ring
55
What parameters are critical to hydride transfer?
Distance and angle of approach of hydride. In NADH, this is 3-3.5Å, also known as van der Waals distance
56
What are the basic steps in fatty acid biosynthesis?
Oxidation -> hydration -> oxidation
57
Describe type I fatty acid synthase
Contain catalytic domains 1 and 2 Found in bacteria, vertebrates and yeast
58
Describe type II fatty acid synthase
Contain discrete polypeptides catalysing each enzymatic step Found in plants and many bacteria
59
How do acyl carrier proteins bring acyl groups to fatty acid synthase?
Its phophoantetheine covalently linked to a serine residue ( S-acyl primed) attacks the acyl group.
60
What is the first step of fatty acid elongation?
Oxidation: uses β-keto ACP reductase NADP+ reduced tetrameric to trigonal planar
61
Describe the β-keto ACP reductase (BKR) active site?
Rossman fold and S...YxxxK motif. Tyr and Lys in the active site
62
Describe Bassica napus ENR (enoyl-acyl carrier protein reductase)
Isoform from developing rapeseed seeds Isolated from chloroplast Tetramer with each subunit being 32.5kDa NADH specific
63
How do the structures of BKR and ENR compare?
High structural similarity despite <20% sequence identity Active site tyrosines do not directly superimpose Phenyl hydroxyl groups are very close in position
64
Outline the ENR mechanism
Hydride transfer from NADH to C3 at a double bond in the acyl substrate Rearrangement forms an enolate anion A proton is donated from tyrosine side chain Tautomerisation from Enol to keto to give a reduced product
65
How is the ENR active site postitioned?
Positioned for the nicotinamide ring to go in
66
What does a G93 to V93 change in enolate reductases (ENR) cause?
Not much change as van der waals forces overlap
67
How can phosphodiester bond hydrolysis be described?
SN2: Substitution Nucleophilic 2 molecules (bimolecular)
68
What is the associative mechanism of phosphodiester bond cleavage?
Water attacks phosphate HOR group leaves Trigonal pyramidal intermediate
69
What is the dissociative mechanism of phosphodiester bond cleavage?
Water molecule sits close to the phosphate Trigonal planar intermediate forms OR group leaves
70
How can RNA phosphodiester bonds self-cleave?
Alkaline conditions Base attacks 2' OH on ribose which attacks 3' end of phosphate
71
What are the key parts of RNA nucleases?
His12 cleaves phosphate group His119 accepts the proton

Y2 Biochemistry (7 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2025 Bold Learning Solutions. Terms and Conditions