Enzymes - Biochemistry
What is an enzyme
1 or more polypeptide chains that form a catalytic active site
What is a Substrate
Molecules that bind to an active site that is complimentary shape to it and undergo a chemical reaction
What is the role of enzymes
Food digestion + Blood Clottingt and blood pressure control+ Immune systme+ Cell processes +Drug breakdown
What is an Anabolic Reaction
Where you go from a smaller reactant to form a larger product
What is Catabolic reaction
Where you go from a larger reactants to a smaller products
What is an interconvention reaction
Where there some reactants form the same amount of products
How do we classify enzymes
Most enzymes usuallt end in -ase and named based on the reaction they catalyse of their product/substrate
Enzyjmes have specific enzyme commission numbers that is amde up from Class, Subclass, Sub-class and Serial Number
What are the enzymes classes
Oxidoreductase that tranfer electrons as H- or H+
Transferase that tranmfer chemical groups from one place to another
Hydrolases They break down bonds with water in a hydrolysis reaction
Lysases in reactions with double bonds
Isomerases used for the transfer of groups within a molecule to form isomers
Ligases that used in the formation of bonds with energy from ATP
Why are enzymes important for the pace of life
Process have to happen quick in order to match the pace of life
2H2O2 --> 2H2O+O2
This needs to happen quickly due to H2O2 is toxic and damges DNA in our cells, usally takes 41 years for this reaxtion to happen and with enzyme take 1 second
Why are Enzymes important for the conditons of life
We have a set body temperature and neutral pH in our body
Means thayt we can thave extreme conditons to speed up reactions so need enzymes to increase rate of reaction
What can effect the rate of reaction
The speed of 1 reaction = The Rate constant K
Number of reactions that take place
How do enzymes increase the number of reactions
Do this by lowering the activation energy so then greater molecules have the activation energy so greater number of sucessful collison so a greater rate of reaction
What is the enzyme Potency
How many times faster a reaction is with an enzyme compared to without the enzyme, can be used to then compare enzymes and rank them based on how good they are at increasing the rate of reaction
What are the advantage of enzymes
They are not used in the reaction
They are very specific due to unique shape of the active site so no side reactions
100% yield
They are controlable so can tunr on/off molecule production when its needed or not needed
How is the active site determined and made
It is controlled by the 3D arrangement of amino acids
It contains binding and catalytic residues and is the source of substrate and reaction specificity
Usaully not all the substrate enter the active site but only small chemical group of the substrate
What is the lock and key mechanism
Only specific substrate have the complimentary shape to the active site so some wont enter the active site
Some may enter but then not bind to the active site
The substrate has to be a complimentary shape and with the binding affinity to bind with active site
What is the induced fit model
The active site will change shape once the substrate has entered and then it will enclose around the substrate
Satrt off bigger to allow substrate to enter then change shape to match substrate and then reaction can take place
What is the reaction specificty determined by
3D arrangement of the residues and the chemical properties of them
May need the catalytoc triad whcih is only 3 amino acids that perfom catalysis need to be in a specific place
What else may be in the active site
Metal co-factors
Co-enzymes which are organic molecules that provide or remove groups from the reaction
Can be hydrogen shuttles during REDOX
Prosthetic groups may also be their
What are enzyme Kinetics
The characterisation of the rates and steps of catalysis done by the enzyme
How to measure enzyme Kinetics
The start point is measuring the change in cocnentration of product over time after adding the enzyme, this can be measured by a spectrophotometer that measure the absorbance change
What is the enzyme kinetics at equilibrium
The rate of the forward and backward reaction are the same so get an overall rate of 0 due to no change in product concentration even with the enzyme working
How to measure the speed of the reaction
The reaction Velocity
The enzyme activity
The specific activity which is used for protein purity
What is the overall rate of reaction dependent on
This depends on rate constants and concentrations, and due to being a reversible reaction both the forward and backward reaction have own rate constant (K)
So at equilibrium the: K of Substrate to product x [S] = K of product to substrate x [P]
How does the additon of an enzyme effect the rate
The enzyme will provide an alternative route from substate to product at a lower activation energy, so then greater rate of reaction so the equilibrium reached sooner.
How to mathmatically calculate the V(Rate) of an enzyme in a reaction
V= Vmax x [S] / Km + [S]
Vmax= The max rate when all enzymes are binded in enzymes substrate complex
Km= Conc of subsrate that gives the half maximum rate and at which all the enzymes are in an enzyme substrate complex
What are he conclusion from an enzyme saturation curve
At the start enzymes are unsaturated so are spare and so bind to substratesa
At the end the enzymes are fuly saturated so no more enzyme binding to substrate so a lower rate of reaction
What is the Michaelis Menten model
E+S <--> ES<--> E + P
It is a 2 step process due to the binding stage first then the catalysing stage second that have different V equation s
Binding= V = Kfrw x [S] and have to work out the Kd = E x S / ES
Catalysing = V = Kcat x [ES]
What are the assumptions of the Michealis - Menten model
ES is a constant concentration
S is greater than E so S is a constant as well
How to work out what Km is
Km = K binding backwards + Kcatalysing /Kbinding forward
What is the shape of the Michealis Menten curve
Is a curved shape that plateu so can be hard to find values on the graph
What does the LineWeaker Burk plot do
It turns the Micheales menten curve to a straight line by taking the reciprocals of the mentn curve values whihc is 1 over them, and the gradient then allows you to find the Kmax and Vmax if you have one or the other
Can use y intercept = 1/Vmax and X intercept = 1/Kmax
What is the bilogical meaning of Vmax
Always low due to stauration of [S] is unusual
Except in ethanol where have the enzyme but due to volume of ehtnaol drunk then enzymes get saturdated so are drunk
Why is Km biologically important
Usuallty high in cells and the [S] often close to the Km
Different substances have differing Km
Important for methanol poisoning due to methanol converted to formaeldehyde by binding to alcohol dehydrogenase but have a higher Km so treatemnt if force feed ethanol that have 10x lower Km so the enzyme bind to the ethanol not the methanol so its not broken down
How to compare enzymes
Turnover number - Kcat = catalytic rate constant
Enzyme efficiency
Enzyme Potency
How does temperature regulayte enzyme activity
This is due to as temp increases from 0 the enzyme activity increases until the optiumum point where it then will denature and activity falls
After the optimum the temperature causes hydrogen bonds and disulphide bonds to break in the active site
How does pH regulate enzymes
Different enzymes work best at different pH and have a short range either side where then higher than the range the enzyme will denature and lower than the range the active site changes charge
How is enzyme concentration effect the activity
Due to a directly proportional the rate to the enzyme concentration
How are enzymes regulated by Covalent bond regulation
Some enzymes need modifying before they work= digestive enzymes
Controlled by cleavage of of peptide chains on zymogen that block its active site
Enzymes may need other enzymes to become active
Can make an enzyme inactive by phosphorylation of the enzyme or remove phosphates to become active by Kinases
How can enzymes be controlled non-covalently
The reversible binding or unbinding of molecules to specific sites to increase or decrease activity
What is Cooperactivity
This is a special type of K type regulation
Where the sustrate binding to one site will increas the binding affinity in another site
This means the [Substrate] directly regulates the enzyme activity
This happnes in multi sub unit enzymes and has a different curved graph
What arev Allosteric enzymes
Have a special binding site for a non substrate molecule and they have a low affinity till another molecule bind to the non-active site leading to then greater affinity enzyme affinity = Allosteric activator
Can get Allosteric inhibitors that lower the enzyme affinity
What are irreversible enzyme inhibitors
Cause a permanent covalent enzyme alteration due to the active site breaks bond in the inhibitor so then they bond together so then substrate unable to bind
Have to resynthesize the enzyme to allow it to work
What are competitive enzymes
Have a similar shape to subsrate so complimentary shape to the active site
Bind to active site and block the substrate fro, bindng so no enzyme activity
Keep Vmax the same and increase Km
What are non-competitive inhibitors
They bind on the enzyme but not the active site, they prevent the enzyme reaction to take place but the enzyme still bind to the subtrate at the active site
Lower Vmax and keep Km the same
Where are uncompetitive inhibitors
They bind to the enzyme substrate complex and increase Vmax but keep Km the same
What is the sequential method of a multiple substrat/product enzyme reaction
Reaction = A + B <--> C+D
Enzyme A + B --> AB --> CD --> C+D
This can be random or orderded
What is the Ping Pong method for multiple enzyme reactions
A + B <--> C + D
Enzyme + A --> EA --> EC --> Enzyme --> EB --> ED --> Enzyme + D
Where covalent bonds are formed to the enzyme during the process
Enzyme is modified after A to C so then can turn B to D
What are general enzyme strategies used for all enzymes
Position the reactant correct of interaction
Distort the reactants to make them less stable
Stabalise the transition state and make reactant want to react
Change environment to favour reaction
What are chemical strategies for specific enzyme reactions
Covalent catalysis- Enzyme react with substrate
Acid/Base Catalysis - active site donate or accept a H+ to get more acidic or basic
Metal ion catalysis- Mix of non covalenent binding and covalent interaction lower the overall eactivation energy
What is proteolysis
When peptide bonds are cleaved by hydrolysis under condtions whihc are 6m HCL for 24 hrs due to peptide bonds are very stable
What are Serine Preoteases
There are 18,000 serine proteases in 40 familes and 12 clans
This is due to gene duplication and divergence to make
Chymotrypsin, Trypsin, Elastase, Thrombin
They have different substrates but do the same peptide bond hydrolysis
What preptide bonds do the serine proteases break
Chymotrypsin cleave large hydrophobic, Trypisn cleave Lysene and Arginene,Elasatse cleave Small neutral amino acids and Trombin cleave ARG-GLY
Due to all have different active sites
How do Serine Proteases work
S1= There is nucleophillic attack on the polypetide carbonly
S2= Formation of intermediate tetrahedral structure that is covalent
S3= Cleavage and the loss of the C-terminal fragment of the polypeptide chain due to an acyl-enzyme
S4= Nucleophillic attack on the Carbonly by water due to H removed from water to make OH- nucleophile
S5= Another tetrahderal intermediate formed with the addition of water
S6= Cleavage and loss of the N-terminus fragment of the polypeptide and enzyme is fully regenerated
What is an oxyanion hole
Act as a stabilsier during tetrahedral covalent intermediate formation
Due to the O can form covalent bond with active site on the NH of the amino acid to stabilise it