B1-4 Enzymes Flashcards
Definition of Enzymes
Biological catalysts that possess the ability to speed up a reaction without being themselves changed at the end of a reaction
How do enzymes work (not the hypotheses ah don’t think so far)
Enzymes increase the rates of chemical reactions by lowering the free energy barrier that separates the reactants and products i.e. activation energy
Definition of Activation energy (Ea)
Minimum amount of energy required to start a chemical reaction
Lowering of Ea
At physiological temperatures, few molecules can overcome Ea barrier and can only do so with the use of a catalyst
Activation energy is lowered by enzyme
More reactant molecules can surmount the energy barrier to reach the transition state to be converted into product molecules
Total energy difference/ free energy change/ Gibbs free energy change between reactant and product molecules remains the same
Properties of Enzymes (6)
Effective in small amounts (chemically unaltered, reusable)
Extremely efficient
High degree of specificity
Denatured by heat and act most efficiently at optimum T
Affected by pH and act most efficiently at optimum pH
Activity can be regulated by activators and inhibitors
Structure of enzymes
Most are globular proteins
Have specific 3D conformation necessary for their action
3D structure must be maintained for enzyme to remain functional
An enzyme can be denatured when bonds holding them in specific 3D conformation are disrupted
4 categories of aa residues
Catalytic aa residues - R groups involved in catalytic activity
Binding aa residues - R groups hold substrate in position via covalent bonds
Structural aa residues - Maintain specific 3D conformation of active site and enzyme as a whole
Non-essential aa residues - No specific function, can be removed or replaced without loss of enzyme’s catalytic function
Cofactor - Inorganic metal ions
Usually small and divalent (charge +2)
Component of active site or allosteric regulator
Bind reversibly to enzyme and act by altering enzyme’s active and/or allosteric sites to facilitate catalytic reaction carried out by enzyme
Cofactor - Coenzyme
Loosely associated with enzyme during reaction
Act as transient carriers of specific functional groups, hydrogens, or electrons
Most are derived from vitamins
Cofactor - Prosthetic Group
Tightly bound to enzyme on permanent basis
Allosteric Enzymes
Alternate between active and inactive form
Have multiple subunits and through conformational changes, binds activators of inhibitors at sites other than active site
How Enzymes lower Ea
Orientate S in close proximity, in correct orientation, to undergo chemical reactions
Strain critical bonds in S molecule(s), allowing S to attain unstable transition state
Provide microenvironment that favours reactions
Lock and Key Hypothesis
There is an exact fit/complementary shape or conformation between S and active site of E
E is viewed as a rigid structure, where only S exactly complementary to conformation of active site are able to bind to active site for catalysis
Explains substrate specificity of enzymes
Induced Fit Hypothesis
Active site conformation is not precisely complementary to that of S before binding
Active site is not a rigid conformation that fits only one type of substrate
Upon binding, active site of E changes conformation slightly to bind S even more firmly so that R groups of catalytic aas at active site are moulded into specific conformation and brought into close proximity to the chemical bonds in S to facilitate catalysis
Further explains group specificity where one enzyme is able to catalyse reactions for a variety of substrates that share similar structural or chemical properties
Rate of enzyme-catalysed reaction and how to measure
Rate is the amount of S which is converted to P by enzymes per unit time
Measured by rate of product formation or substrate usage
Competitive Inhibitor
Structurally similar to S, compete with S for binding to active site
Although it is not acted upon by E, it remains bound to the active site and prevents S binding to active site
Initial rate reduced, but both reactions reach same Vmax
Requires longer time in presence of inhibitor to reach same Vmax
Km higher for inhibited
Why increase in S conc reduces effect of competitive inhibition
Because S and inhibitor are in direct competition for E’s active sites
Greater proportion of S - Greater chances of out-competing inhibitor - Rate of reaction almost equivalent to Vmax can be attained
Final amount of P formed same as S continues to be converted by E molecules unaffected by inhibitor
Non-competitive Inhibitor
Bears no structural resemblance to substrate, does not compete with S for active site
Binds to part of E that is not active site
Binding of inhibitor alters 3D conformation of E and active site
E molecule no longer has active site complementary in conformation to S
Hence S does not bind to E active site and no E-S complex can be formed
Why increase in S conc reduces effect of non-competitive inhibition
Even when substrate concentration is very high, initial rate of reaction does not reach same Vmax as uninhibited reaction
Binding of non-con to site other than active site causes change in 3D conformation of enzyme active site that prevents S from binding
Why non-competitive inhibitor has lower Vmax
Certain proportion of E molecules are rendered inactive
As S and inhibitor are not in direct competition for same site, increase in S concentration has no effect on inhibition
Km remains unchanged as affinity of enzyme for substrate remains unaffected
Final amount of P formed same as S continues to be converted by E molecules unaffected by inhibitor
Why non-competitive inhibitor has lower Vmax
Certain proportion of E molecules are rendered inactive
As S and inhibitor are not in direct competition for same site, increase in S concentration has no effect on inhibition
Km remains unchanged as affinity of enzyme for substrate remains unaffected
Final amount of P formed same as S continues to be converted by E molecules unaffected by inhibitor
Allosteric Regulation
Regulation of an E by binding of molecules (regulators: activators, inhibitors) at an allosteric site i.e. site other than active site
Allosteric enzyme structure
Most allosterically regulated E are composed of 2 or more polypeptide chains - Multi-subunit E
Each subunit has its own active site and allosteric sites are usually located where subunits are joined
Allosteric Inhibition
Binding of activator to active site induces favourable conformation change in active site of all subunits of enzyme
This significantly amplifies response of E to substrates. The amplification results in sudden steep rise in rate of reaction
Cooperativity
Binding of 1 S molecule to an active site of a multimeric enzyme triggers same favourable conformation change in active site of all other subunits of E
Likewise amplifies response of E to S - 1 S molecule primes E to accept additional S molecules
Reversible vs Irreversible inhibition
Inhibitor binds via weak non-covalent (hydrogen bonds, hydrophobic interactions) VS covalent bonds
Reversible - Effect is temporary, inhibitor can be easily removed and causes no permanent damage
Irreversible - Inhibitor causes permanent damage to E as it is unable to carry out catalytic activity
Factors affecting Rate of Enzymatic reaction
Substrate Concentration
Enzyme Concentration
Temperature
pH
Kcat / Turnover number
Maximum number of molecules of S that an enzyme can convert to P per catalytic site per unit time
General steps to explaining rate of reaction
Frequency of effective collisions
Rate of formation of enzyme-substrate complexes
Rate of product formation
Effect of low T
Enzymes are inactivated
Effect of T beyond optimum T
Decrease in rate of reaction despite increasing frequency of collisions (decreasing stability)
Thermal agitation of E molecule disrupts hydrogen bonds, ionic bonds, and other non-covalent interactions that stabilise the specific 3D conformation of protein molecule
Loss in 3D conformation of E and that of its active site so that there is no longer a complementary fit with S
E said to be denatured and lose its catalytic function
Frequency of effective collisions decreases… (typical stuff)
Changes in pH can affect E activity by…
Altering ionic charge of acidic and basic R groups
Low pH - More H+ ions available to neutralise negative charges in enzyme
Higher pH - Less H+ ions available to…
Change in ionisation of aas disrupts ionic bonds / hydrogen bonds that maintain 3D conformation of enzyme, denaturing enzyme
Care about effect on all aa residues except non-essential ones
Optimum pH
Enzymes work most efficiently
Rate of reaction at max, intra-molecular bonds are intact and conformation of active site is ideal for binding, therefore frequency of EC is highest with largest amount of ES complexes formed
At pH values only slightly above and below optimum, many enzymes will have marked decline in E activity as 3D conformation is altered and affinity for substance is correspondingly decreased
Optimum pH is usually pH of environment in which the enzyme normally functions
Vmax
Max rate at which E is able to perform the reaction in a specific concentration of E and excess S
Km
S concentration that allows an enzyme-catalysed reaction to proceed at half of Vmax
Represents affinity of E for particular S
Low value + High affinity
Feedback / end-product inhibition
Cellular control mechanism in which an enzyme’s activity is inhibited by the enzyme’s end product
