L7 - Review of enzymes & enzyme reactions

Question 1

What are enzymes?

Answer 1

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

Question 2

How do enzymes accelerate reactions?

Answer 2

By facilitating the formation of the transition state

The formation of an enzyme-substrate complex is the first step in enzymatic catalysis

Question 3

Where does catalysis take place?

Answer 3

At the active site of the enzyme

Question 4

Are enzymes generally protein?

Answer 4

Nearly all known enzymes are proteins

However, it has been shown that certain RNA molecules can act as enzymes (ribozymes)

Question 5

What is equilibrium?

Answer 5

The state in which both reactants and products are present at concentrations which have no further tendency to change with time

Question 6

Example of when enzymes may transform energy from one form to another

Answer 6

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

Question 7

Substrate specificity in enzymes

Answer 7

Enzymes catalyse a single chemical reaction or a set of closely related reactions but can vary in degree of substrate specificity (Eg. proteolytic enzymes)

Question 8

Examples of enzymes that catalyse proteolysis (hydrolysis of the peptide bond)

Answer 8

Subtilisin
Trypsin
Thrombin

Question 9

Subtilisin, trypsin & thrombin all catalyse proteolysis, but how are their substrate specificities different?

Answer 9

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

Question 10

What drives enzyme specificity?

Answer 10

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

Question 11

What are cofactors?

Answer 11

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

Question 12

Whats the difference in a cofactor being tightly or loosely bound?

Answer 12

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

Question 13

Are metals important cofactors?

Answer 13

Yes

> 25% of all enzymes need specific metal ions to function

Question 14

What are the 6 major classes of enzymes?

Answer 14

Oxidoreductases 
Transferases. 
Hydrolase 
Lyases 
Isomerases 
Ligases

Question 15

What is the transition state?

Answer 15

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

Question 16

What is the activation energy?

Answer 16

The difference in free energy between the substrate and transition state is called the activation energy

Question 17

What is the function of enzymes in relation to the AE?

Answer 17

The function of an enzyme is to lower the activation energy by facilitating the formation of the transition state

Question 18

Indirect evidence for the formation of an ES complex

Answer 18

Kinetic evidence

Spectroscopic evidence

Question 19

Kinetic evidence for the formation of an ES complex

Answer 19

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

Question 20

Spectroscopic evidence for the formation of an ES complex

Answer 20

The spectroscopic characteristics of many enzymes and substrates change on mixing – indicates the formation of an ES complex

Question 21

Direct evidence for the formation of an ES complex

Answer 21

Structural evidence

X-ray crystallography has provided high-resolution structures of many enzymes with substrate or substrate analogues bound to their active sites

Question 22

What is the active site of an enzyme?

Answer 22

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

Question 23

Effect of specific amino acids in the active site

Answer 23

Amino acids in the active site provide complementary shape, charge and hydrophilic or hydrophobic characteristics required for substrate binding and catalysis

Question 24

How are substrates bound to the active site?

Answer 24

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

Question 25

H bonds between enzyme and substrate

Answer 25

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

Question 26

Electrostatic attraction between enzyme and substrate

Answer 26

Negatively or positively charged amino acids (Glu or Asp –ve; Arg or Lys +ve at neutral pH) interact with opposite charge group on substrate

Question 27

Van der Waals forces between enzyme and substrate

Answer 27

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

Question 28

Hydrophobic interactions between enzyme and substrate

Answer 28

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

Question 29

Lock & key model for enzyme-substrate binding

Answer 29

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

Question 30

Induced fit model for enzyme-substrate binding

Answer 30

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.