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Protein Structure & Function > Catalytic Mechanisms I > Flashcards

Catalytic Mechanisms I Flashcards

1
Q

Name the 6 catalytic mechanisms

A

Acid-base
Covalent
Metal ion
Electrostatic
Proximity & orientation
Preferential transition state binding

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2
Q

What does catalysis result in?

A

Lowers the free energy of activation (ΔG#)

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3
Q

What is an example of an acid-base catalyst?

A

RNase A = cleaves RNA molecule at the phosphodiester bond

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4
Q

What reaction does RNase A catalyse and how?

A

Cleaves RNA molecule at the phosphodiester bond

Hydrolysis reaction

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5
Q

What is the intermediate and second substrate of the RNase reaction?

A

2,3-cyclic nucleotide = intermediate
H2O = substrate

Hydrolysis reaction = cleaves intermediate, leading to final product

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6
Q

What special group does RNase A have at its active site?

A

Two histidine residues, which have imidazole groups

Imidazole groups can function as both base and acid

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7
Q

What does the Hendeerson-Hasselbalch equation say?

A

pH = pKa + log [A-]/[HA]

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8
Q

What happens when acid and base conc are equal?

A

pH = pKa

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9
Q

What does pKa tell us?

A

pKa value is one method used to indicate the strength of an acid.

pKa is the negative log of the acid dissociation constant or Ka value.

A lower pKa value indicates a stronger acid

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10
Q

What does covalent catalysis involve?

A

TRANSIENT formation of catalyst-substrate covalent bond

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11
Q

Give an example of covalent catalysis

A

Nucleophilic attack

Nucleophile (enzyme) attacks electrophile (substrate) and transient covalent bond formation results

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12
Q

Define nucleophiles and electrophiles

A

Nucleophile = electron rich
Electrophiles = electron poor

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13
Q

Give examples of nucleophiles

A

electron RICH
S
O
N

AND imidazole groups

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14
Q

What reaction does pyruvate decarboxylase catalyse and what is needed?

A

Pyruvate to acetaldehyde (alcohol fermentation)

Requires COFACTOR = thiamine pyrophosphate (TPP)

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15
Q

What is the role of thiamine pyrophosphate in decarboxylation?

A

TPP has thiazolium ring (nucleophile) attacks carbonyl C of pyruvate

CO2 leaves = generates carbanion
Protonation of carbanion

Elimination of TPP to form acetyldehyde and regerate active enzyme

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16
Q

What is the difference between metal-activated enzymes and metalloenzymes?

A

Metal-activated enzymes = loosely bind metal ions

Metalloenzymes = contain tightly bound transition metal ions

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17
Q

What is the role of Mg2+ in relation to ATP?***

A

Shield negative charge

Can form 6 coordinate bonds = so can be involved in interaction with enzymes

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18
Q

What is the role of Zn2+ in liver alcohol dehydrogenase?

A

Role in orientation/polarization of ETHANOL (substrate)

OH- is attracted to Zn2+ so it sits in the active site in the correct orientation

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19
Q

What is the role of Zn2+ in carbonic anhydrase?

A

CO2 + H2O&raquo_space;> H2CO3

Zinc is tetrahedrally coordinated to 3 His side chains and then water molecule

Zinc helps to polarize the water

20
Q

What is the His residue not bonded to Zn2+ and its ROLE?***

A

His 64 part of active site

21
Q

How does electrostatic catalysis work?

A

Increases reaction rate by arrangement of charge distribution around active site

Stabilizes the transition state of the catalyzed reaction

May so guide polar substrates to their binding sites = increase rate of reaction

22
Q

What effect do enzymes have on proximity and orientation?

A

Bing substrates into contract w catalytic group/other substrates

Bind substrate in right orientation

Freeze out translational and rotational motions of substrates/catalytic groups

23
Q

Give an example of enzyme that effects proximity and orientation

A

Adenylate kinase = part of the nucleoside monophosphates (NMP)

24
Q

What form do NMPs exist in within the cell?

A

Mg2+ - NMP

Without metal ion = NO enzyme activity

25
Q

What is the structure of adenylate kinase?

A

P-loop ad lid domain

26
Q

What conformational change occurs when adenylate kinase binds ATP?

A

P-loop closes down and interacts with beta P group

Gamma P group is thus positioned next to binding site of second substrate

27
Q

What is preferential transition state binding?

A

Enzymes bind the transition state of the reaction with greater affinity than its substrate

28
Q

Give an example of an enzyme with preferential transition state binding

A

Proline racemase

29
Q

What are the 5 approaches to determine enzyme catalytic mechanisms?

A

Kinetic studies

Intermediates

X-ray crystallography

Chemical modifications of side chains

Site-directed mutagenesis

30
Q

What is chymotrypsin?

A

Digestive enzyme classified as a serine protease, primarily found in the small intestine.

It catalyzes the hydrolysis of peptide bonds in proteins

31
Q

How was chymotrypsin’s intermediate used to determine catalytic mechanism?

A

Acyl-enzyme intermediate exists for very short time

Can manipulate conditions to isolate intermediate and do X_ray crystallography study

Found out that side chain of Ser195 = acylated

32
Q

What is chymotrypsin catalytic triad?

A

His 57
Asp 102
Ser 195

33
Q

What are the 3 serine proteases?

A

Chymotrypsin
Trypsin
Elastase

34
Q

What residues are the serine proteases specific for?

A

Chymotrypsin = bulk hydrophobic
Trypsin = positively charged residues
Elastase = small neutral residues

35
Q

Describe the substrate binding pockets of serine proteases

A

Chymotrypsin = small side chains to fit the bulky residues

Trypsin = negative residues (Asp) to attract positive residues

Elastase =

36
Q

What is used in affinity labelling?

A

Substrate analog

Bearing reactive group that specifically binds at the enzyme active site

AND forms stable covalent bond with enzyme

37
Q

What is affinity labelling used for?

A
38
Q

What is TPCK?

A

Affinity label for chymotrypsin

39
Q

How does TPCK affect chymotrypsin?

A

TPCK will inactivate chymotrypsin because it will make covalent bonds at active site

40
Q

How do you inhibit the labelling of an enzyme?

A

Adding substrate or competitive inhibitor will inhibit the labelling of TPCK

41
Q

What evidence proves that a modified side chain is at the active site?

A

Linear relationship between extent of modification and inactivation

Addition of substrate must protect against inactivation

42
Q

What are the pre-requisites for site-directed mutagenesis?

A

Gene/ORF coding for the protein is available

Suitable system for expression of the gene/ORF

43
Q

What are the steps of site-directed mutagenesis

A

3D structure is known or can be predicted

Using structure = select residues that may be important in catalysis

Mutate these residues and determine the effect on activity of the enzyme

44
Q

What are the two types of site-directed mutagenesis?

A

Conserved mutation = replace with amino acid with similar properties

Non-conserved mutation = replace with amino acid with different properties

45
Q

What bonds does chymotrypsin cleave?

A

Both ester and amide bonds

46
Q

What is the reaction of p-Nitrophenylacetate and chymotrypsin?

A

p-nitrophenylacetate + chymotrypsin

Products = pNP and acyl-enzyme intermediate

Split the acyl-enzyme intermediate = acetate and enzyme

47
Q

Describe the two kinetic phases

A

Burst phase = rapid formation of p-NP and correlates to amount of active enzyme

Steady-state = pNP is generated at a reduced but constant rate

Protein Structure & Function (11 decks)

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