Download ENZYMES

ENZYMES
A protein with catalytic properties due to its
power of specific activation
© 2007 Paul Billiet ODWS
Reaction pathway
Chemical reactions need initial input of energy
= THE ACTIVATION ENERGY
During this part of the reaction the molecules
are said to be in a transition state.
© 2007 Paul Billiet ODWS
Enzymes Modify Chemical
Reactions





Reactions with enzymes are faster
Reactions with enzymes are more specific
Enzymes increase rate of reactions without
increasing temperature.
They do this by lowering activation energy.
They create a new reaction pathway “a short cut”
© 2007 Paul Billiet ODWS
(edited by ckelly 2014)
An enzyme controlled pathway

Enzyme controlled reactions proceed 108 to 1011 times faster
than corresponding non-enzymic reactions.
© 2007 Paul Billiet ODWS
Enzyme structure



Enzymes are
proteins
They have a
globular shape
A complex 3-D
structure
© 2007 Paul Billiet ODWS
Human pancreatic amylase
© Dr. Anjuman Begum
The active site


© H.PELLETIER, M.R.SAWAYA
ProNuC Database
© 2007 Paul Billiet ODWS
A pocket or space of an
enzyme that fits the
substrate.
The shape and the
chemical environment
inside permits a
chemical reaction to
proceed
Cofactors



Additional non-protein
molecules needed by
some enzymes to help
the reaction
Cofactors that are bound
and released easily are
called coenzymes
Many vitamins are
coenzymes
Nitrogenase enzyme with Fe, Mo and ADP cofactors
Jmol from a RCSB PDB file © 2007 Steve Cook
© 2007 Paul Billiet ODWS
H.SCHINDELIN, C.KISKER, J.L.SCHLESSMAN, J.B.HOWARD, D.C.REES
STRUCTURE OF ADP X ALF4(-)-STABILIZED NITROGENASE COMPLEX AND ITS
IMPLICATIONS FOR SIGNAL TRANSDUCTION; NATURE 387:370 (1997)
The substrate



The reactants are changed by the enzyme
Enzymes are specific to their substrates
Specificity is determined by the active site
© 2007 Paul Billiet ODWS
The Lock and Key Hypothesis





Fit between substrate and active site of enzyme is exact
Temporary structure called enzyme-substrate complex forms
Products have a different shape from the substrate
Once formed, they are released from the active site
Enzyme is free to act on another substrate
© 2007 Paul Billiet ODWS
The Lock and Key Hypothesis
S
E
E
E
Enzymesubstrate
complex
Enzyme may
be used again
P
P
© 2007 Paul Billiet ODWS
Reaction coordinate
The Lock and Key Hypothesis


This explains enzyme specificity
This explains the loss of activity when
enzymes denature
© 2007 Paul Billiet ODWS
What does an ES Complex do?
-
-
-
holds substrate out of aqueous solution
holds substrate in specific orientation, close to Transition State to allow
reaction to occur
reduces ability of free rotation & molecular collisions with non-reactive
atoms
allows an altered local environment: changes ionic strength, pH, adds or
removes H-bonds to substrate
Terminology
Many enzymes require a non-protein component for activity:

cofactor: small inorganic ions... mostly metal ions: Cu (cytochrome
oxidase), Mg (kinases), Fe (catalase, peroxidase)

coenzymes: small non-protein but organic compounds
Coenzyme A: acyl transfer
Flavins: redox reaction
NAD+ (NADP+): redox reactions
Vitamins: derivatives of B vitamins (B1, B2, B6, B12), niacin, folic acid,
riboflavin

prosthetic group: tightly bound large complex organic molecules, (heme)
Holoenzyme vs apoenzyme (apoprotein)
The Induced Fit Hypothesis


Some proteins change their shape when binding
substrate
The bonds of the substrate are stretched to make the
reaction easier (lowers activation energy)
© 2007 Paul Billiet ODWS
The Induced Fit Hypothesis
Hexokinase (a) without (b) with glucose substrate
http://www.biochem.arizona.edu/classes/bioc462/462a/NOTES/ENZYMES/enzyme_mechanism.html

This explains the enzymes that can react with a
range of substrates of similar types
© 2007 Paul Billiet ODWS
Factors affecting Enzymes




substrate concentration
pH
temperature
inhibitors
© 2007 Paul Billiet ODWS
Substrate concentration: Non-enzymic reactions
Reaction
velocity
Substrate concentration

The increase in velocity is proportional to the
substrate concentration
© 2007 Paul Billiet ODWS
Substrate concentration: Enzymic reactions
Vmax
Reaction
velocity
Substrate concentration


Faster reaction but it reaches a saturation point when all the
enzyme molecules are occupied.
If you alter the concentration of the enzyme then Vmax will
change too.
© 2007 Paul Billiet ODWS
The effect of pH
Optimum pH values
Enzyme
activity
Trypsin
Pepsin
1
© 2007 Paul Billiet ODWS
3
5
7
pH
9
11
The effect of temperature
Q10
Enzyme
activity
0
© 2007 Paul Billiet ODWS
10
20
30
40
Temperature / °C
Denaturation
50
Inhibitors




Reduce the rate of enzymatic reactions.
Usually specific and work at low
concentrations.
Block the enzyme but they do not usually
destroy it.
Many drugs and poisons are inhibitors of
enzymes in the nervous system.
© 2007 Paul Billiet ODWS
DHFR —
Dihydrofolate
reductase
•Converts dihydrofolate into tetrahydrofolate (THF)
by the addition of a hydride from NADPH
•THF is a methyl (CH3) group shuttle required for
synthesis of essential molecules
- nucleotides
- amino acids
22
During reaction
NADPH becomes NAD+
23
Enzyme Assay
24
How to define enzyme activity?

1 unit ACTIVITY= International unit (IU)
amount enzyme which converts 1 μmole substrate per min at 25oC
 e.g. IU= 10 μmole/min

1 unit SPECIFIC ACTIVITY
# IU of enzymatic activity per mg of total protein present
 e.g. 10 μmole/min/mg protein or 10 IU/mg protein
Tools of enzymology-1
Spectroscopic techniques (structure and reactivity in solution)

Optical (circular dichroism, UV-visible, fluorescence)

Vibrational (infrared, Raman)
Electrochemical methods (kinetic analysis)

Potentiometric techniques

Conductometry
Enthalpimetry (microcalorimetry)

Very sensitive and free of interference
Radiochemical methods

Far more sensitive than photometric ones but...
Tools of enzymology-2
X-ray crystallography

First crystallized enzyme, urease (J. Sumner, in 1926)  crystals are
proteins and their dissolution led to enzymatic activity

Within 20 years: >130 enzymes crystals documented

3-D structure of a protein, myoglobin, was deduced (Kendrew, 1957)
Multidimentional nuclear magnetic resonance (NMR) and X-ray
crystallography are now commonly used:


to explain the mechanistic details of enzyme catalysis
to design new ligands
Molecular Biology

Clone and express enzymes in foreign hosts (overexpression 
purification and characterization of enzymes occuring naturally in minute
quantity)

Manipulate the a.acid sequence (site-directed mutagenesis and deletional
mutagenesis  chemical groups in ligand binding)
The effect of enzyme inhibition
Irreversible inhibitors: Combine with the
functional groups of the amino acids in the
active site, irreversibly.
Examples: nerve gases and pesticides,
containing organophosphorus, combine with
serine residues in the enzyme acetylcholine
esterase.

© 2007 Paul Billiet ODWS
The effect of enzyme inhibition
Reversible inhibitors: These can be washed
out of the solution of enzyme by dialysis.
There are two categories.

© 2007 Paul Billiet ODWS
The effect of enzyme inhibition
1. Competitive: These
compete with the
substrate molecules for
the active site.
The inhibitor’s action is
proportional to its
concentration.
Resembles the substrate’s
structure closely.
© 2007 Paul Billiet ODWS
E+I
Reversible
reaction
EI
Enzyme inhibitor
complex
The effect of enzyme inhibition
Fumarate + 2H++ 2e-
Succinate
Succinate dehydrogenase
CH2COOH
COOH
CHCOOH
CH2
CH2COOH
COOH
Malonate
© 2007 Paul Billiet ODWS
CHCOOH
The effect of enzyme inhibition
2. Non-competitive: These are not influenced by the
concentration of the substrate. It inhibits by binding
irreversibly to the enzyme but not at the active site.
Examples
 Cyanide combines with the Iron in the enzymes
cytochrome oxidase.
 Heavy metals, Ag or Hg, combine with –SH groups.
These can be removed by using a chelating agent such
as EDTA.
© 2007 Paul Billiet ODWS
Applications of inhibitors



Negative feedback: end point or end product
inhibition
Poisons snake bite, plant alkaloids and nerve
gases.
Medicine antibiotics, sulphonamides,
sedatives and stimulants
© 2007 Paul Billiet ODWS