L7 - Review of enzymes & enzyme reactions Flashcards
What are enzymes?
They are catalysts of biological systems
Enzymes accelerate reactions but they do not alter the equilibrium
Enzymes may transform energy from one form to another
Enzymes are specific
Many enzymes require cofactors
Enzymes are classified on the basis of the types of reactions they catalyse
How do enzymes accelerate reactions?
By facilitating the formation of the transition state
The formation of an enzyme-substrate complex is the first step in enzymatic catalysis
Where does catalysis take place?
At the active site of the enzyme
Are enzymes generally protein?
Nearly all known enzymes are proteins
However, it has been shown that certain RNA molecules can act as enzymes (ribozymes)
What is equilibrium?
The state in which both reactants and products are present at concentrations which have no further tendency to change with time
Example of when enzymes may transform energy from one form to another
In oxidative phosphorylation the electron-motive force is converted into a proton-motive forces by a series of membrane bound enzymes
This proton-motive force is then converted into phosphoryl transfer potential (the generation of ATP by ATP synthase).
Enzymes may then use the chemical-bond energy of ATP in many ways
Substrate specificity in enzymes
Enzymes catalyse a single chemical reaction or a set of closely related reactions but can vary in degree of substrate specificity (Eg. proteolytic enzymes)
Examples of enzymes that catalyse proteolysis (hydrolysis of the peptide bond)
Subtilisin
Trypsin
Thrombin
Subtilisin, trypsin & thrombin all catalyse proteolysis, but how are their substrate specificities different?
Subtilisin - will cleave any peptide bond with little regard to the identity of adjacent side chains
Trypsin - is quite specific and catalyses the splitting of peptide bonds only on the carboxyl side of lysine and arginine (+ively charged) residues
Thrombin - is more specific than trypsin and catalyses the hydrolysis of Arg-Gly bonds in a particular sequence
What drives enzyme specificity?
The specificity of an enzyme is due to the precise interaction of the substrate with the enzyme
This precision is a direct result of the intricate three-dimensional (3D) structure of the enzyme-protein
What are cofactors?
These enable enzymes to carry out reactions that cannot be performed by the standard set of 20 amino acids
Apo-enzyme + cofactor = holo-enzyme
Cofactors divided into two groups:
- Coenzymes (small organic molecules)
- Metals
Whats the difference in a cofactor being tightly or loosely bound?
If cofactors are tightly bound, they are also called prosthetic groups
If loosely associated, they are more like co-substrates, that bind and are released like substrates and products
Are metals important cofactors?
Yes
> 25% of all enzymes need specific metal ions to function
What are the 6 major classes of enzymes?
Oxidoreductases Transferases. Hydrolase Lyases Isomerases Ligases
What is the transition state?
The transition state has a higher free energy (∆G) than either S or P
Transition state is a transitory molecule that is no longer substrate but is not yet product
Least stable and most seldom occupied species on reaction pathway as highest free energy
What is the activation energy?
The difference in free energy between the substrate and transition state is called the activation energy
What is the function of enzymes in relation to the AE?
The function of an enzyme is to lower the activation energy by facilitating the formation of the transition state
Indirect evidence for the formation of an ES complex
Kinetic evidence
Spectroscopic evidence
Kinetic evidence for the formation of an ES complex
At constant concentration of enzyme, the rate of reaction increases with increasing substrate concentration until it reaches a maximal velocity – hyperbolic curve
Non-catalysed reactions do not show this saturation effect.
Saturation suggests the formation of a discrete ES complex where at a sufficiently high substrate concentration all of the catalytic sites are filled and so reaction rate cannot increase
Spectroscopic evidence for the formation of an ES complex
The spectroscopic characteristics of many enzymes and substrates change on mixing – indicates the formation of an ES complex
Direct evidence for the formation of an ES complex
Structural evidence
X-ray crystallography has provided high-resolution structures of many enzymes with substrate or substrate analogues bound to their active sites
What is the active site of an enzyme?
The active site of an enzyme is the region that binds the substrate(s) + cofactor (if any) and contains the residues (catalytic groups) that directly participate in the making and breaking of bonds.
The interaction of the enzyme and substrate at the active site promotes the formation of the transition state
The active site is a three-dimensional cleft/crevice/pocket formed by groups that often come from different parts of the amino acid sequence
Active sites are unique microenvironments
Effect of specific amino acids in the active site
Amino acids in the active site provide complementary shape, charge and hydrophilic or hydrophobic characteristics required for substrate binding and catalysis
How are substrates bound to the active site?
Substrates are bound by multiple weak reversible interactions (non-covalent bonds)
- H bonds
- Electrostatic attraction
- Van der Waals forces
- Hydrophobic interactions
+ INTERACTIONS BETWEEN METAL/CO-FACTORS & SUBSTRATE
H bonds between enzyme and substrate
Hydrogen atom bound to electronegative atom (O or N in proteins) is polar
Interacts with polar groups on substrate
Lots of H bonds between E & S
Electrostatic attraction between enzyme and substrate
Negatively or positively charged amino acids (Glu or Asp –ve; Arg or Lys +ve at neutral pH) interact with opposite charge group on substrate
Van der Waals forces between enzyme and substrate
All atoms have slight dipole caused by rotation of electrons round nucleus.
Weak attraction when two atoms close together – needs complementary shapes
Very weak but lots of them
Hydrophobic interactions between enzyme and substrate
Much more energetically favourable for two hydrophobic surfaces (ie. protein and substrate) to interact together than with water in bulk solvent
Involves nonpolar amino acids such as phenylalanine, leucine etc
Lock & key model for enzyme-substrate binding
Initially proposed by Emil Fisher (1890) that enzymes and substrates are an exact match for each other
Problem: this model explains enzyme specificity, but not the stabilisation of the transition state that enzymes achieve
Induced fit model for enzyme-substrate binding
The active site can assume a shape that is complementary to that of the substrate only after the substrate has bound – induced fit (Koshland 1958)
This model proposes that the initial interaction between enzyme and substrate is relatively weak, but that these weak interactions rapidly induce conformational changes in the enzyme that strengthen binding to the transition state and stabilise it, so reducing the activation energy to reach it.
