Enzyme- Multi-substrate Reactions

Question 1

What is an example of a multi-substrate reaction?

Answer 1

ATP+ Creative forms ADP + Phosphocreatine

Question 2

How man yreactions are multi-substrate?

Answer 2

Nearly two thirds

Question 3

When is MMK not valid?

Answer 3

If two substrates can vary in concentration

Question 4

What are the steps to MMK of multi-substrate reactions?

Answer 4

Fixation of S1, varying concentration of S2, LB determination of apparent Km and apparent S2, repeat process with S2

Question 5

What are the different reaction mechanisms possible?

Answer 5

Both substrates bind at once forming a ternary complex
Substrates bind and leave where catalysis occurs after first substrate binds

Question 6

Ternary complex

Answer 6

This is a protein complex consisting of three different molecule that are bound together

Question 7

Displacement Reactions

Answer 7

These are products normally consisting of two pjhases, the metal or a compound and the oxide or chloride subject to reductant

Question 8

What is an example of Displacemnet Reactions?

Answer 8

Lactate Dehydrgeonase is sequential where S1 and S2 bind sequentially and are replaced by two different products
Aspartate Aminotransferase

Question 9

Lactate Dehydrogenase

Answer 9

This is an enzyme important in anaerobic metabolic pathways converting pyruvate to lactae

Question 10

Aspartate Aminotransferase

Answer 10

This is a transaminase enzyme that catalyses the conversion of aspartate and a-ketoglutarate to oxaloacetate and glutamate

Question 11

What is the function of AAT

Answer 11

Catalyses reversible transfer of an amino group between aspartate and AKH

Question 12

What is the mechanisitc basis of AAT function?

Answer 12

Containing of a cofactor pyridoxal 5’-phosphate acting as a carrier for amino group

Question 13

What is the process of AAT mechanism?

Answer 13

S1 binds forming schiff base intermediate with transfer of substrate amino group to the cofacto

Question 14

Schiff Base

Answer 14

This is the imino bonds formed by nucleophillic attack of amine to aldehyde

Question 15

How is oxaloacetate formed in AAT?

Answer 15

Rearragnement of schiff base to oxaloacetate regenerating enzyme resting state

Question 16

What is the second reaction in AAT?

Answer 16

AKG binds after S1 release forming new schif base with amino group transfer to the AKG with second product formation

Question 17

How does S2 2relate in a LWP?

Answer 17

Increasing S2 decreases slope in a sequential displacement reaction where apparent Vmax increases and Km decreases

Question 18

Chymotrypsin

Answer 18

This is a serine protease produced by the pancrease hydrolysing the peptide bonds of tryptophan, leucine, tyrosine and phenylalanine

Question 19

What is the mechanistic basis of Chymotyrpsin?

Answer 19

Cleaves peptide bonds selectively on CTD of large hydrophobic amino acid

Question 20

How is Chymotrypsin a DDR?

Answer 20

First displacement is nucleophilic attack from hydroxyl of serine forming intermediary with peptide bond cleavage/fragment formation
Abstration of a base of an active site serine residue

Question 21

How can catalytic residues be identified?

Answer 21

Irreversible inhibitors covalently binding to the enzyme

Question 22

What are the three types of irreversible inhibitor?

Answer 22

Group-specific Reagents
Affinity Labels
Suicide Inhibitors

Question 23

Group-Specific Reagents

Answer 23

These are enzyme inhibitors that can covalently bind to a particular amino acid residue on the enzyme and irreversibly modify it

Question 24

Affinity Labels

Answer 24

This is a technique for labelling the binding site of proteins by virtue of a ligand analog to which a chemically reactive or photoreactive group has been attacehd

Question 25

Suicide Inhibtors

Answer 25

These act by forming stable acyl-enzyme complexes, which fragment and deacylate very slowly

Question 26

What is the catalytic triad of Chymotrypsin?

Answer 26

Ser195, His57, Asp102

Question 27

What is the mechanism of the catalytic triad?

Answer 27

His57 polarisation of the OH group of the Ser195, this serine becoming hihgly reactive ion

Question 28

How does Asp102 assist in the triad?

Answer 28

Positioning His57 and countering the positive charge

Question 29

What enzymes is the catalytic triad found in?

Answer 29

Chymotrypsin
Subtilisin
Wheat CarboxyPeptidase

Question 30

Wheat Carboxypeptidase II

Answer 30

This is a zinc metalloenzyme catalysing the hydrolysis of the neurotransmitted N-acetyl-L-aspartyl-L-glutamate to another residue

Question 31

How can importance of active site residues be elucidated?

Answer 31

Site-directed MMutagenesis

Question 32

Site-directed Mutagenesis

Answer 32

This is a technique where DNA can be modified at a specific nucleotide location causing a predetermined amino acid change