Enzyme- Multi-substrate Reactions Flashcards
What is an example of a multi-substrate reaction?
ATP+ Creative forms ADP + Phosphocreatine
How man yreactions are multi-substrate?
Nearly two thirds
When is MMK not valid?
If two substrates can vary in concentration
What are the steps to MMK of multi-substrate reactions?
Fixation of S1, varying concentration of S2, LB determination of apparent Km and apparent S2, repeat process with S2
What are the different reaction mechanisms possible?
Both substrates bind at once forming a ternary complex
Substrates bind and leave where catalysis occurs after first substrate binds
Ternary complex
This is a protein complex consisting of three different molecule that are bound together
Displacement Reactions
These are products normally consisting of two pjhases, the metal or a compound and the oxide or chloride subject to reductant
What is an example of Displacemnet Reactions?
Lactate Dehydrgeonase is sequential where S1 and S2 bind sequentially and are replaced by two different products
Aspartate Aminotransferase
Lactate Dehydrogenase
This is an enzyme important in anaerobic metabolic pathways converting pyruvate to lactae
Aspartate Aminotransferase
This is a transaminase enzyme that catalyses the conversion of aspartate and a-ketoglutarate to oxaloacetate and glutamate
What is the function of AAT
Catalyses reversible transfer of an amino group between aspartate and AKH
What is the mechanisitc basis of AAT function?
Containing of a cofactor pyridoxal 5’-phosphate acting as a carrier for amino group
What is the process of AAT mechanism?
S1 binds forming schiff base intermediate with transfer of substrate amino group to the cofacto
Schiff Base
This is the imino bonds formed by nucleophillic attack of amine to aldehyde
How is oxaloacetate formed in AAT?
Rearragnement of schiff base to oxaloacetate regenerating enzyme resting state
What is the second reaction in AAT?
AKG binds after S1 release forming new schif base with amino group transfer to the AKG with second product formation
How does S2 2relate in a LWP?
Increasing S2 decreases slope in a sequential displacement reaction where apparent Vmax increases and Km decreases
Chymotrypsin
This is a serine protease produced by the pancrease hydrolysing the peptide bonds of tryptophan, leucine, tyrosine and phenylalanine
What is the mechanistic basis of Chymotyrpsin?
Cleaves peptide bonds selectively on CTD of large hydrophobic amino acid
How is Chymotrypsin a DDR?
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
How can catalytic residues be identified?
Irreversible inhibitors covalently binding to the enzyme
What are the three types of irreversible inhibitor?
Group-specific Reagents
Affinity Labels
Suicide Inhibitors
Group-Specific Reagents
These are enzyme inhibitors that can covalently bind to a particular amino acid residue on the enzyme and irreversibly modify it
Affinity Labels
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
Suicide Inhibtors
These act by forming stable acyl-enzyme complexes, which fragment and deacylate very slowly
What is the catalytic triad of Chymotrypsin?
Ser195, His57, Asp102
What is the mechanism of the catalytic triad?
His57 polarisation of the OH group of the Ser195, this serine becoming hihgly reactive ion
How does Asp102 assist in the triad?
Positioning His57 and countering the positive charge
What enzymes is the catalytic triad found in?
Chymotrypsin
Subtilisin
Wheat CarboxyPeptidase
Wheat Carboxypeptidase II
This is a zinc metalloenzyme catalysing the hydrolysis of the neurotransmitted N-acetyl-L-aspartyl-L-glutamate to another residue
How can importance of active site residues be elucidated?
Site-directed MMutagenesis
Site-directed Mutagenesis
This is a technique where DNA can be modified at a specific nucleotide location causing a predetermined amino acid change
