Catalytic Mechanisms I

Question 1

Name the 6 catalytic mechanisms

Answer 1

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

Question 2

What does catalysis result in?

Answer 2

Lowers the free energy of activation (ΔG#)

Question 3

What is an example of an acid-base catalyst?

Answer 3

RNase A = cleaves RNA molecule at the phosphodiester bond

Question 4

What reaction does RNase A catalyse and how?

Answer 4

Cleaves RNA molecule at the phosphodiester bond

Hydrolysis reaction

Question 5

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

Answer 5

2,3-cyclic nucleotide = intermediate
H2O = substrate

Hydrolysis reaction = cleaves intermediate, leading to final product

Question 6

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

Answer 6

Two histidine residues, which have imidazole groups

Imidazole groups can function as both base and acid

Question 7

What does the Hendeerson-Hasselbalch equation say?

Answer 7

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

Question 8

What happens when acid and base conc are equal?

Answer 8

pH = pKa

Question 9

What does pKa tell us?

Answer 9

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

Question 10

What does covalent catalysis involve?

Answer 10

TRANSIENT formation of catalyst-substrate covalent bond

Question 11

Give an example of covalent catalysis

Answer 11

Nucleophilic attack

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

Question 12

Define nucleophiles and electrophiles

Answer 12

Nucleophile = electron rich
Electrophiles = electron poor

Question 13

Give examples of nucleophiles

Answer 13

electron RICH
S
O
N

AND imidazole groups

Question 14

What reaction does pyruvate decarboxylase catalyse and what is needed?

Answer 14

Pyruvate to acetaldehyde (alcohol fermentation)

Requires COFACTOR = thiamine pyrophosphate (TPP)

Question 15

What is the role of thiamine pyrophosphate in decarboxylation?

Answer 15

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

CO2 leaves = generates carbanion
Protonation of carbanion

Elimination of TPP to form acetyldehyde and regenerate active enzyme

Question 16

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

Answer 16

Metal-activated enzymes = loosely bind metal ions

Metalloenzymes = contain tightly bound transition metal ions

Question 17

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

Answer 17

Shield negative charge

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

Question 18

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

Answer 18

Role in orientation/polarization of ETHANOL (substrate)

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

Question 19

What is the role of Zn2+ in carbonic anhydrase?

Answer 19

CO2 + H2O > H2CO3

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

Zinc helps to polarize the water

Question 20

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

Answer 20

His 64 part of active site

Question 21

How does electrostatic catalysis work?

Answer 21

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

Question 22

What effect do enzymes have on proximity and orientation?

Answer 22

Bring substrates into contact w catalytic group/other substrates

Bind substrate in right orientation

Freeze out translational and rotational motions of substrates/catalytic groups

Question 23

Give an example of enzyme that effects proximity and orientation

Answer 23

Adenylate kinase = part of the nucleoside monophosphates (NMP)

Question 24

What form do Nucleoside Monophosphate exist in within the cell?

Answer 24

Mg2+ - NMP

Without metal ion = NO enzyme activity

Question 25

What is the structure of adenylate kinase?

Answer 25

P-loop and lid domain

Question 26

What conformational change occurs when adenylate kinase binds ATP?

Answer 26

P-loop closes down and interacts with beta P group Gamma P group is thus positioned next to binding site of second substrate

Question 27

What is preferential transition state binding?

Answer 27

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

Question 28

Give an example of an enzyme with preferential transition state binding

Answer 28

Proline racemase

Question 29

What are the 5 approaches to determine enzyme catalytic mechanisms?

Answer 29

Kinetic studies Intermediates X-ray crystallography Chemical modifications of side chains Site-directed mutagenesis

Question 30

What is chymotrypsin?

Answer 30

Digestive enzyme classified as a serine protease, primarily found in the small intestine. It catalyzes the hydrolysis of peptide bonds in proteins

Question 31

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

Answer 31

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

Question 32

What is chymotrypsin catalytic triad?

Answer 32

His 57 Asp 102 Ser 195

Question 33

What are the 3 serine proteases?

Answer 33

Chymotrypsin Trypsin Elastase

Question 34

What residues are the serine proteases specific for?

Answer 34

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

Question 35

Describe the substrate binding pockets of serine proteases

Answer 35

Chymotrypsin = small side chains to fit the bulky residues Trypsin = negative residues (Asp) to attract positive residues Elastase =

Question 36

What is used in affinity labelling?

Answer 36

Substrate analog Bearing reactive group that specifically binds at the enzyme active site AND forms stable covalent bond with enzyme

Question 37

What is affinity labelling used for?

Question 38

What is TPCK?

Answer 38

Affinity label for chymotrypsin

Question 39

How does TPCK affect chymotrypsin?

Answer 39

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

Question 40

How do you inhibit the labelling of an enzyme?

Answer 40

Adding substrate or competitive inhibitor will inhibit the labelling of TPCK

Question 41

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

Answer 41

Linear relationship between extent of modification and inactivation Addition of substrate must protect against inactivation

Question 42

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

Answer 42

Gene/ORF coding for the protein is available Suitable system for expression of the gene/ORF

Question 43

What are the steps of site-directed mutagenesis

Answer 43

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

Question 44

What are the two types of site-directed mutagenesis?

Answer 44

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

Question 45

What bonds does chymotrypsin cleave?

Answer 45

Both ester and amide bonds

Question 46

What is the reaction of p-Nitrophenylacetate and chymotrypsin?

Answer 46

p-nitrophenylacetate + chymotrypsin Products = pNP and acyl-enzyme intermediate Split the acyl-enzyme intermediate = acetate and enzyme

Question 47

Describe the two kinetic phases

Answer 47

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