Biological Chemistry

Lecture 12

Enzyme kinetics Part 1

 Learning outcomes
You will be able to:

•Identify lock and key versus induced mechanisms

•Recognise the major categories of enzyme activities

•Appreciate that enzymes lower activation energy

•Appreciate that enzymes do not affect the equilibration point

only the time taken to achieve it

•Appreciate that specific amino acids play key roles in catalysis

 Some Basic Terminology

Apoenzyme: Is an inactive enzyme, activation of the enzyme occurs upon binding of an

organic or inorganic cofactor

Holoenzyme: An apoenzyme together with its cofactor. A Holenzyme is complete

and catalytically active. Most cofactors are not covalently bound but instead
are tightly bound

Cofactor: Two categories 1. Metal ions 2. Coenzymes (non protein organic complexes –
sometimes referred to as a prosthetic group or chromophore

Substrate: The material or substance on which an enzyme acts

Lock and key model of enzyme-substrate binding

In this model, the active site of the unbound enzyme is

Complementary in shape to the substrate
 Induced-fit model of enzyme-substrate binding

In this model, the enzyme changes shape on substrate binding. The active
site forms a shape complimentary to the substrate only after the substrate
has been bound.
 Carbonic anhydrase

One of the fastest enzymes known: hydrating 10 6 molecules of CO2 PER SECOND
Class of Enzyme Type of reaction Examples
Oxidoreductase Oxidation-reduction Succinate
dehydrogenase
Transferases Group transfer Hexokinase
Hydrolases Hydrolysis reactions Trypsin
(transfer of
functional groups to
water)
Lyases/Dehydratases Breakage of C-C Aldolase/enolase
bonds in organic
molecules/ Addition
or removal of groups
to form double
bonds
Isomerases Isomerization Aconitase
(intramolecular
group transfer)
Ligases Ligation of two Pyruvate carboxylase
substrates at the
expense of ATP
hydrolysis
1. Oxidoreductases
2. Transferases
 3. Hydrolases

Many macromolecules are degraded by this type of reaction

Enzyme specificity: Trypsin cleaves on the carboxyl side of
arginine and lysine residues
4. Lyases and dehydratases

Lyases

dehydratase
 5. Isomerases

Role: preparation of molecule for subsequent reactions

6. Ligases
 Assaying enzymes

• Enzymes assayed by activity

Continuous assay e.g. a spectrophotometric assay-often in
Vis part of spectrum. One of the reactants forming a chromophore
complex with lmax often in 340-600nm region e.g. DTNB and CoA;
generation of NADH. Chemiluminescence using luciferase.
Specificity of reaction has to be validated because of multiple competing
activities. E.g.using acyl-CoA as substrate.

Discontinuous assay. Determinations made at specific time points e.g.

5s, 10s, 20s, 30s, 60s. Could be spectrophotometric as above or could
be based on isotope incorporation 14C or 3H or 32P.

• Structural proteins: purifcation can be followed by immunotechniques-

antibody binding assays. Enzyme-linked immunosorbent assays (ELISA)
and Western blotting on SDS-PAGE.
 Enzyme assays

Formation of NADH can be measured at 340nm

Enzymes decrease the activation energy

Glyceraldehyde 3-phosphate dehydrogenase:

a case study
Enzymes decrease the activation energy
Energy transformation steps of glycolysis

1,3-BPG is an acyl phosphate with a high

phosphoryl transfer potential

Reaction proceeds in two stages

Step 1 oxidation Go’ = -12Kcal mol-1
Step 2 acyl phosphate formation Go’ = +12Kcal mol-1
Structure of glyceraldehyde 3-phosphate
dehydrogenase
Catalytic mechanism of glyceraldehyde 3-phosphate
dehydrogenase

Cysteine residue of enzyme reacts with G3-P

Hydride ion transfer to NAD+
NAD+ replaces NADH first

Positive charge on NAD+

may polarise thioester to facilitate
attack by Pi
 Summary

•Lock and key and induced fit are two enzymatic

mechanisms

•Six types of reaction are identified internationally

•Enzymes lower activation energy but do not affect the

equilibration point only the time taken to achieve it

•Specific amino acids play key roles in catalysis