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CHAPTER 4 : ENZYMES - Coggle Diagram
CHAPTER 4 : ENZYMES
ENZYME ARE CATALYST
- increase the rate of reaction
- not consumed by the reaction
- act repeatedly to increase the rate of the reaction
- very specific which only 1 particular reactant with 1 enzyme
WHY USE ENZYME?
- accelerate and control the rate of vitally important biochemical reactions
- greater reaction specificity
- milder reaction conditions
- capacity for regulation
- enzyme are agents of metabolic functions
- metabolites have many potential pathways
- enzyme make the desired one most favourable
- doesnt change the equilibrium constant of a reaction
- doesnt alter the standard free energy change of a reaction
= amount of energy consumed or liberated in the reaction
= quantity that determines if a reactionis energetically favourable
= thermodynamically favourable or spontaneous (free energy change is negative)
- doesnt change thermodynamics
ENZYME DECREASE THE ACTIVATION ENERGY OF REACTION
- activation energy is the energy required to start a reaction
- the lesser the energy needed to start the reaction, the faster the reaction will go
- provide a different mechanism and pathway for reaction through. lower energy pathway
- enzyme bind to substrate molecules to form an enzyme-substrate complex
E+S > ES
- formation of the transition state complex where the bound substances is neither product nor reactant
ES > ES*
- formation of the enzyme-product complex
ES* > EP
- release of product
EP > E+P= once the product is released, the enzyme is unchanged and can carry out another reactionsE+S > ES > ES* > EP > E+P
ACTIVE SITE
- Have specificity
- can discriminate among the possible substrate molecules
- others recognise a functional group
- some only recognize one type of molecules
- Active site relatively small 3D region within the enzyme
- typically it is a small cleft or crevice on a large protein
- substrate bind in active site by weak non-covalent interactions
- H bond
- hydrophobic interactions
- ionic interactions
- these interaction hold the substrat in the proper orientation fo most effective catalysis
- each non-covalent interactions provides energy to stabilize the transition state which can use to lower the activation energy and stabilize the transition state complex (ES*)
2 MODELS FOR THE ENZYME AND SUBSTRATE INTERACTIONS
- LOCK AND KEY MODEL
- substrate (key) fits into a perfectly shaped space in the enzyme (lock)
- there are a lot of similarity of shape of the enzyme and the shape of the substrate
- highly stereospecific
- implies a very rigid inflexible active site
- site is preformed and rigid
- INDUCED FIT MODEL
- takes ino account of flexibility of proteins
- a substrate fits into a general shape in the enzyme causing the enzyme to change shape (conformation) close but not perfect fit
- change in protein configuration leads to near perfect fit of substrate with enzyme
ENZYME LOWER THE ACTIVATION ENERGY OF THE REACTION
- by stabilizing the transition state
- it puts the molecules in close proximity to react so increase the local concentration of reactans
- put molecules in correct oritentation which reactant are not only near each other on enzyme, theyre oriented in optimal position to react, making it possible to always collide in the correct orientation.
Proximity and orientation
- called transition state theory
= enhance the formation of and stabilize the highly energetic transition state
= transition state binds more tightly that substrate or product
- enzyme bind tightly to the transition state species
= binding energy helps reach and stabilize the transition state > lower the activation energy > increase the rate of reaction
- transition state stabilization accomplished through close complementary in shape and charge between the active site and the transition state
= reaction proceed bond are broken and new ones formed transforming S > P
= following catalysis the product no longer fit the active site and is released
*Transition-state analog enzyme model
- enzyme bind the transition state structure more tightly than the substrate
- enzyme active site are complementary not to the substrate but to the transition state
- binding of the substrate result in the distortion of the substrate and enzyme in way that makes the chemical reaction easier
ENZYME KINETICS
- enzyme acitivity can be assayed in many ways
= dissappearance of substrate
= appearance of product
eg :
~ appearance of coloured product made from an uncoloured substrate
~ appearance of a UVabsorbent product made from a non-UV-absorbent substrate
~ appearance of radioactive product made from radioactive substrate VELOCITY
- the velocity (V) of an enzyme catalyzed reaction is dependent upon substrate concentration (S)
- according to the graph V vs S it is often hyperbolic and called Michaelis-Menten plot
- 2 analogies :
a. Toll plaza (with 5 booths)
= rate at which cars can get through the booths is not affected by the number of waiting cars only by the available number of toll attendants
b. paper airplane example
QUANTITATIVE EXPRESSION OF ENZYME BEHAVIOR
- Michaelis-menten eq describe the kinetic behavior of many enzymes
E+S > ES > E+P
a. reverse reaction P > S is not considered because the eq describe intial rates when [P] is near zero
b. ES complex is. steady intermediate beacause it is produced and broken down at the same rateMichaelis-Menten eqV = Vmax [S] _________
Km + [S]
V = reaction rate (velocity) at a substrate concentration [S]
Vmax = maximum rate that can be observed in the reaction substrate is present in excess (enzyme can be saturated)Km (Michaelis constant)
- it is a constant that is related to the affinity of the enzyme for the substrate units in ters of concentrations
- it measure the ES binding (how well it binds)
Km = K-1 + K2 _________
K1
~ small Km means tight binding, large Km means weak bindingKm = [S] at 1/2 Vmaxindicates how efficiently an enzyme select its substrate and converts to productssmall Km so the enzyme achieve maximal catalytic effeciencyV = Vmax / 2
TURNOVER NUMBER (kcat) (CATALYTIC CONSTANT
- indicate how fast ES complex proceeds to E+P
- number of catalytic cycles that each active site undergoes per unit time
turnover number = Kcat = Vmax/ [Et][Et] = total enzyme concentrationKcat/Km = catalytic efficiency
- reflect both binding and catalytic event indicates how the velocity varies according to how oftern the enzyme and substrate combine
- best value represent enzyme ability to convert substrate to product
-
ENZYME INHIBITIONInhibitors
- interfere with the action of an enzyme
- decrease the rates of their catalyst
- great focus at many drug company as to develop comound to prevent certain disease due to enzymatic acitvity
eg : AIDS and HIV protease inhibitors= HIV protease essential for processing of proteins in virus
= without these proteins, viabl viruses cannot be released to cause further infection
- can be reversible and irreversible
a. irreversible inhibitors
= covalently modified after interaction with inhibitors
= derivatized enzyme is no longer a catalyst as it lost enzymatic activity
= orignial activity cannot be regenered
= called suicide inhibitorsb. reversible inhibitors
= bind to enzyme and are subsequently released
= leave enzyme in original condition
= can be distinguished by their kinetics of inhibition
= three subclasses :
- competitive inhibitors
- non-competitive inhibitors
- uncompetitive inhibitors
COMPETITIVE INHIBITORS
- shape and structure of inhibitors is very similar to substrate
- inhibitors mimic substrate and fit into active site
- physically block substrate from bind to active site
- kinetics of their inhibition
= Km increase
= Vmax stay the same
eg : HIV protease inhibitors such as Squinavir and Viracept*Succinate dehydrogenase
- transition state analogs are compound that resemble the transition state of a catalyzed reaction
- TS analogs bind much stronger to the enzyme than simple substrate or product analogs
NON-COMPETITIVE INHIBITORS
- inhibitors bind to a site other than the active site
- binding cause a change in the structure of the enzyme so that it cannot catalyze a reaction
- effect of non-competitive inhibition cannot be overcome by increasing [S]
- kinetics of inhibition
= Km stay the same
= Vmax decrease
MIXED INHIBITION
- affect both substrate binding and Vmax
= Km increase
= Vmax decrease
- mixed inhibition is when the inhibitor bind to the enzyme at a location distinct from the substrate binding site
- it is the same concept as non-competitive inhibition but the binding of inhibitor will affect the binding affinity
- binding affinity for the substrate decrease when the inhibitor is present
UNCOMPETITIVE INHIBITORS
- inhibitor bind to a site other than active site but only when substrate is bound (bind to ES complex)
- distorts active site which prevent reaction from occuring
- kinetics of inhibition
= Km decrease
= Vmax decrease
- the effect of uncompetitive inhibition cannot be overcome by increasing [S]
IRREVERSIBLE INHIBITORS
- inhibitors covalently modify the active site permanent inhibition
eg : aspirin, nerve gas
COENZYMES
- some enzyme require additonal component for activity
- used at the active site of the enzyme
- not covalently bound to the enzyme
- can be small organic molecules or metal ions
- lossely attached to apoenzyme and separated easily by dialyss
- many are structurally related to vitamins
- they are regenerated for further reactions
Eg : NAD+, FAD, TPP and Lipoic acidPROSTHETIC GROUPS
“ coenzyme that are covalently bound to an enzyme and therefore are always present”
- why vitamins acts as coenzyme?
= group transfer agents
= carrying electrons
= chemical group such as acyl groups, methyl group depends on the coenzyme
eg : vitamin A,D,E and K
- vitamin niacin (nicotinic acid) in NADH & NADPH (Vitamin B3)
- help in oxidation reduction reaction with enzyme
- biotin
- a prosthetic coenzyme that catalyzes carboxyl-group transfer
- ATP-dependent carboxylation reactions
- Vitamin E (alpha-tocopherol)
- important lipid-soluble antioxidant that helps protect polyunsaturated fatty acids in membrane phospholipids from oxidative damage
- picks up electron from lipid free radical species
- Riboflavin (vitamin B2) in FAD and FMN
- important electron carriers for a wide variety of biological processes
REGULATION OF ENZYME ACTIVITY
- to make an enzyme more or less active
- regulation is necessary to control the rates of reactions and to properly synchronize all of the metabolic reactions in the cell
- modify the intrinsic properties of the enzyme
a. non-covalent interaction
= bind regulatory molecules reversibly eg : protein, lipid and small moleculesb. reversible covalent modifications
= phosphorylation of serine, threonine or tyrosine
= methylation of glutamate residues (used in bacteria as food sensor)
- induction/repression
- change rat of enzyme synthesis or degradation of the enzyme
- change cellular distribution of the enzyme
- creation or reduction of disulfide bonds
IRREVERSIBLE COVALENT MODIFICATIONS
- addition of fatty acids and fatty acids derivatives (isoprenylation, acylation and palmitoylation)
2. addition of sugars to Asparangine (Glycosylation)3. proteolytic cleavage
- ZYMOGENS inactive precursor to an enzyme which activated by cleavage of a specific peptide bond
eg : = proteolytic enzyme trypsin and chymotrypsin
= initially synthesized as trysinogen and chymotrypsinogen which both inactive condition
= but it is activated by cleavage of specific peptide bonds when they are needed for the digestive properties