ENZYMES
Crystal structure of trypsin
http://en.wikipedia.org/wiki/Trypsin
Function of enzymes
they are special proteins produced by living cells
• they are catalysts → increase the rate of chemical reactions
and decrease the activation energy of the reaction
• the action of most enzymes is very specific – substrate and
reaction specifity
●
Enzyme catalysis
Enzymes E are able to specifically
bind the reactants (their
substrates S) at the active site →
complex E-S (transition state, ↓
activation energy) → destruction of
complex E-S to products P and E
http://en.wikipedia.org/wiki/Enzyme
Characteristics of enzymes
• Intracellular enzymes
• Extracellular enzymes
• Simple enzymes – only protein structure
• Complex enzymes = protein structure + cofactor
Cofactors are nonprotein compounds.
Cofactor can be:
1) inorganic element: Zn2+, Mn2+, Mg2+, Fe2+, Cu2+, ….
2) organic molecule
a) coenzymes are slightly bound to the enzyme, undergo a chemical
change and are released: NAD(P)+, FAD, coenzyme Q,..
b) prosthetic groups are tightly bound to the enzyme and remain
associated with enzyme during reaction: heme, …
Coenzymes
NAD+ ↔ NADH + H+
nicotinamide adenine dinucleotide
FAD ↔ FADH2
flavin adenine dinucleotide
(vit. B2 = riboflavin)
Other examples: coenzyme A, coenzyme Q, tetrahydrofolate, thiamine
diphosphate (vit. B1 = thiamine)
http://web.indstate.edu/thcme/mwking/vitamins.html
Prosthetic groups
Biotin (vit. H)
Heme
Another example: pyridoxal phosphate (derivate of vitamine B6)
http://web.indstate.edu/thcme/mwking/vitamins.html
Nomenclature of enzymes
1) The first discovered enzymes were named according to their
source:
name of enzyme + suffix -in
Pepsin is found in the gastric juice (Greek pepsis = digestion).
2) Enzymes were named according to their substrate:
name of substrate + suffix –ase
Lipase catalyzes the hydrolysis of lipids.
Urease catalyzes the hydrolysis of urea.
3) In 1961 International Union of Biochemistry recommended that
enzymes be systematically classified according to the general type
of reaction they catalyze → 6 major classes.
Each enzyme has a EC number (four-digit number)
Lactate dehydrogenase has the EC number 1.1.1.27
Classification of enzymes
1. Oxidoreductases catalyze redox reactions
alcohol dehydrogenase
oxidases, oxygenases, peroxidases, catalase
2. Transferases catalyze the transfer of functional groups between
donors and acceptors
aminotransferases, kinases
3. Hydrolases catalyze the hydrolytic cleavage of substrates
peptidases, proteases, lipases, α-amylase
4. Lyases (synthases) catalyze non-hydrolytic and non-oxidation cleavage
or synthesis of molecules (removing/addition of the small molecule from/to
substrate)
carboxylases/decarboxylases, hydratases/dehydratases
5. Isomerases catalyze intramolecular changes in substrate molecules
epimerases, mutases
6. Ligases (synthetases) catalyze synthetic reactions where 2 molecules are
joined at 1 molecule, synthesis requires an energy (ATP)
polymerases
Enzyme kinetics
The Michaelis-Menten model
Michaelis constant KM corresponds to the substrate concentration [S]
at which velocity V is half of the maximum velocity Vmax (when v = ½
Vmax). An enzyme with a high affinity for its substrate has a low KM
value.
KM = mol/L
http://en.wikipedia.org/wiki/Enzyme
Enzyme kinetics
• Lineweaver-Burk plot provides a useful graphical
method for analysis of the Michaelis-Menten
equation:
• taking the reciprocal gives
Figure was found at http://en.wikipedia.org/wiki/Lineweaver-Burk_diagram
Enzymatic activity
Reaction rate is expressed as a change in concentration per unit
time (mol/L / s).
For enzyme-catalyzed reaction: substrate turnover per unit time
is commonly used:
● Unit: katal (kat) = mol of substrate / s
kat and nkat are used in medicine
● International unit: IU = μmol of substrate / min
1 kat = 6
x
107 U
Factors that influence enzyme activity
• Concentration
of substate
The rate of an enzymatic reaction increases as the
substrate concentration increases until a limiting rate is
reached.
•
Concentration of enzyme
Enzyme concentration is much lower than the
concentration of substrate. The rate of an enzymecatalyzed reaction is directly dependent upon the enzyme
concentration.
Temperature
Most enzymes of warm-blooded animals have temperatures optimum
of about 37 oC. Protein structure of enzymes is denatured by heat
(above 55 oC)
• Hydrogen ion concentration (pH)
Extreme values of pH (low/high) cause denaturation of protein.
Optimum pH of enzyme is a narrow pH range.
Optimal pH for pepsin is 2.0 in the stomach, and for trypsin is 8.0 in
small intestine.
●
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/E/Enzymes.html
●
Inhibitors
Some chemical compounds can act as enzyme inhibitors.
Enzyme inhibition:
a) irreversible
b) reversible
Irreversible inhibition
Irreversible inhibitors react with enzyme and form a covalent
adduct with protein or metal ion.
HCN inactives iron-containing enzymes because it binds to Fe2+ in
heme. HCN blocks cellular respiration (cytochrome c oxidase).
The nerve gases inhibit transmission in nerve system because they
block specific enzymes (tabun, sarin).
Competitive inhibition
Competitive inhibitor I „competes“ with a
substrate S for binding at enzyme´s active
site.
plus inhibitor
Vmax value is unchanged
KM value is elevated (it is necessary to add
more S to reach the original enzyme activity)
no inhibitor
Noncompetitive inhibition
Inhibitor I binds to the enzyme site that is
distinct from the active site.
I binds with an equal affinity to the free
enzyme and to the E-S complex
plus inhibitor
Vmax is decreased
(↓ concentration of an active enzyme)
no inhibitor
KM value is unchanged
Uncompetitive (anticompetitive)
inhibition
Inhibitor I binds only to the E-S complex
Vmax and KM values are decreased
Enzyme regulation
• Allosteric enzymes
• Covalent modification of enzymes
a) phoshorylation/dephosphorylation
b) limited proteolysis
Zymogenes (proenzymes) are nonactive forms of enzymes.
They are activated by cleavage of peptide from their molecule.
e. g. trypsinogen → trypsin + hexapeptide
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/E/Enzymes.html
Diagnostic applications of enzymes
The measurement of enzyme activity in body fluids (plasma, serum)
has become an important tool in medical diagnosis. Under normal
conditions the concentrations of enzymes is low in blood.
An abnormally high level of a particular enzyme in the blood often
indicates specific tissue damage (hepatitis, myocardial infarction,....)
Some important enzymes for clinical diagnosis:
Enzyme assayed
Organ or tissue damaged
-amylase (AMS)
pancreas
alkaline phosphatase (ALP)
bone, liver
creatine kinase (CK)
muscle, heart
lactate dehydrogenase (LD)
heart, liver
alanine aminotransferase (ALT)
liver
aspartate aminotransferase (AST)
heart, liver
Example:
normal (physiological) activity of ALT in blood:
up to 0.73 kat/L
Activity of ALT in serum during acute virus hepatitis is 50x higher
than normal activity!
ISOENZYMES
Some enzymes have variants called isoenzymes that catalyze the
same chemical reaction, but isoenzymes have different physicalchemical properties. Organ localization can be different in case of
isoenzymes.
Example:
lactate dehydrogenase (LD) has 5 isoenzymes: LD1 – LD5.
LD isoenzymes are found in skeletal muscle, liver, heart, kidney,
erytrocytes. Isoenzymes can be separated by electrophoresis.