Transcript Allosteric enzymes

Introduction. Structure, properties and biological functions of
proteins. Methods of secretion and purification. Peptides.
Complex proteins, their biological role. Structure and physicalchemical properties of enzymes
Structure of enzymes
Enzymes
Complex or holoenzymes (protein part
and nonprotein part – cofactor)
Apoenzyme (protein
part)
Simple (only protein)
Cofactor
Prosthetic groups
Coenzyme
-usually small inorganic
molecule or atom;
-large organic
molecule
-usually tightly bound to
apoenzyme
-loosely bound to
apoenzyme
Example of prosthetic group
Metalloenzymes
contain firmly
bound metal ions
at the enzyme
active sites
(examples: iron,
zinc, copper,
cobalt).
Example of metalloenzyme: carbonic
anhydrase contains zinc
Coenzymes
• Coenzymes act as group-transfer reagents
• Hydrogen, electrons, or groups of atoms can be
transferred
Coenzyme classification
(1) Metabolite coenzymes - synthesized from
common metabolites
(2) Vitamin-derived coenzymes - derivatives of
vitamins
Vitamins cannot be synthesized by mammals, but
must be obtained as nutrients
Examples of metabolite coenzymes
ATP can donate
phosphoryl group
ATP
S-adenosylmethionine
donates methyl groups
in many biosynthesis
reactions
S-adenosylmethionine
5,6,7,8 - Tetrahydrobiopterin
Cofactor of nitric oxide synthase
Vitamin-Derived Coenzymes
• Vitamins are required for coenzyme synthesis
and must be obtained from nutrients
• Most vitamins must be enzymatically
transformed to the coenzyme
• Deficit of vitamin and as result correspondent
coenzyme results in the disease
NAD+ and NADP+
• Nicotinic acid (niacin) an nicotinamide are precursor of
NAD and NADP
• Lack of niacin causes the disease pellagra
NAD and
NADP are
coenzymes
for
dehydrogenases
FAD and FMN
• Flavin adenine dinucleotide (FAD) and Flavin
mononucleotide (FMN) are derived from riboflavin (Vit B2)
• Flavin coenzymes are involved in oxidation-reduction
reactions
FMN (black), FAD (black/blue)
Thiamine Pyrophosphate (TPP)
• TPP is a
derivative of
thiamine (Vit B1)
• TPP participates
in reactions of:
(1) Oxidative
decarboxylation
(2) Transketolase enzyme
reactions
Pyridoxal Phosphate (PLP)
• PLP is derived from Vit B6 family of vitamins
PLP is a coenzyme for enzymes catalyzing reactions involving amino
acid metabolism (isomerizations, decarboxylations, transamination)
Enzymes active sites
Substrate usually is relatively small
molecule
Enzyme is large protein molecule
Therefore substrate binds to specific
area on the enzyme
Active site – specific region in the
enzyme to which substrate molecule is
bound
Active site of lysozym consists of six amino acid
residues which are far apart in sequence
Active site contains functional groups (-OH, -NH, -COO etc)
Binds substrates through multiple weak interactions
(noncovalent bonds)
Theories of active site-substrate
interaction
Fischer theory (lock and key model)
The enzyme active site (lock) is able to accept only a
specific type of substrate (key)
Koshland theory (induced-fit model)
The process of substrate binding induces specific
conformational changes in the the active site region
Properties of Enzymes
Specificity of enzymes
1.Absolute – one enzyme acts only on one substrate
(example: urease decomposes only urea; arginase
splits only arginine)
2.Relative – one enzyme acts on different
substrates which have the same bond type
(example: pepsin splits different proteins)
3.Stereospecificity – some enzymes can catalyze
the transformation only substrates which are in
certain geometrical configuration, cis- or trans-
Sensitivity to pH
Each enzyme has maximum activity at a particular pH
(optimum pH)
For most enzymes the optimum pH is ~7 (there are
exceptions)
Sensitivity to temperature
Each enzyme has
maximum activity at a
particular
temperature (optimum
temperature)
-Enzyme will
denature above 4550oC
-Most enzymes have
temperature
optimum of 37o
Naming of Enzymes
Common names
are formed by adding the suffix –ase to the name
of substrate
Example:
- tyrosinase catalyzes oxidation of tyrosine;
- cellulase catalyzes the hydrolysis of cellulose
Common names don’t describe the chemistry of the
reaction
Trivial names
Example: pepsin, catalase, trypsin.
Don’t give information about the substrate,
product or chemistry of the reaction
The Six Classes of Enzymes
1. Oxidoreductases
• Catalyze oxidation-reduction reactions
- oxidases
- peroxidases
- dehydrogenases
2. Transferases
• Catalyze group transfer reactions
3. Hydrolases
• Catalyze hydrolysis reactions where water
is the acceptor of the transferred group
- esterases
- peptidases
- glycosidases
4. Lyases
• Catalyze lysis of a substrate, generating a
double bond in a nonhydrolytic, nonoxidative
elimination
5. Isomerases
• Catalyze isomerization reactions
6. Ligases (synthetases)
• Catalyze ligation, or joining of two substrates
• Require chemical energy (e.g. ATP)
Kinetic properties of enzymes
Study of the effect of substrate concentration on the rate of reaction
Enzyme inhibition
In a tissue and cell different chemical agents
(metabolites, substrate analogs, toxins,
drugs, metal complexes etc) can inhibit the
enzyme activity
Inhibitor (I) binds to an enzyme and prevents
the formation of ES complex or breakdown it to
E+P
Reversible and irreversible
inhibitors
Reversible inhibitors – after combining with
enzyme (EI complex is formed) can rapidly
dissociate
Enzyme is inactive only when bound to inhibitor
EI complex is held together by weak,
noncovalent interaction
Three basic types of reversible inhibition:
Competitive, Uncompetitive, Noncompetitive
Reversible inhibition
Competitive inhibition
•Inhibitor has a structure similar to the substrate
thus can bind to the same active site
•The enzyme cannot differentiate between the
two compounds
•When inhibitor binds, prevents the substrate
from binding
•Inhibitor can be released by increasing substrate
concentration
Competitive inhibition
Example of
competitive
inhibition
Benzamidine
competes with
arginine for binding
to trypsin
Noncompetitive inhibition
• Binds to an enzyme site different from the active
site
• Inhibitor and substrate can bind enzyme at the same
time
•Cannot be overcome by increasing the substrate
concentration
Uncompetitive inhibition
• Uncompetitive inhibitors bind to ES not to free E
• This type of inhibition usually only occurs in
multisubstrate reactions
Irreversible Enzyme Inhibition
very slow dissociation of EI complex
Tightly bound through covalent or noncovalent
interactions
Irreversible inhibitors
•group-specific reagents
•substrate analogs
•suicide inhibitors
Group-specific reagents
–react with specific R groups of amino acids
Substrate analogs
–structurally similar to the substrate for the
enzyme
-covalently modify active site residues
Suicide inhibitors
•Inhibitor binds as a substrate and is initially
processed by the normal catalytic mechanism
•It then generates a chemically reactive
intermediate that inactivates the enzyme
through covalent modification
•Suicide because enzyme participates in its
own irreversible inhibition
Regulation of enzyme
activity
Methods of regulation of enzyme
activity
•
•
•
•
Allosteric control
Reversible covalent modification
Isozymes (isoenzymes)
Proteolytic activation
Allosteric enzymes
Allosteric enzymes have a second regulatory
site (allosteric site) distinct from the active
site
Allosteric enzymes contain more than one
polypeptide chain (have quaternary structure).
Allosteric modulators bind noncovalently to
allosteric site and regulate enzyme activity via
conformational changes
Example of allosteric enzyme - phosphofructokinase-1
(PFK-1)
• PFK-1 catalyzes an early step in glycolysis
• Phosphoenol pyruvate (PEP), an
intermediate near the end of the pathway
is an allosteric inhibitor of PFK-1
PEP
Phosphorylation reaction
Dephosphorylation reaction
Usually phosphorylated enzymes are
active, but there are exceptions
(glycogen synthase)
Enzymes taking part in phosphorylation are called protein kinases
Enzymes taking part in
dephosphorylation are called
phosphatases
Isoenzymes (isozymes)
Some metabolic processes are regulated by enzymes that
exist in different molecular forms - isoenzymes
Isoenzymes - multiple forms of an enzyme which
differ in amino acid sequence but catalyze the same
reaction
Isoenzymes can differ in:
 kinetics,
 regulatory properties,
 the form of coenzyme they prefer and
 distribution in cell and tissues
Isoenzymes are coded by different genes
Example: lactate dehydrogenase (LDG)
Lactate + NAD+
pyruvate + NADH + H+
Lactate dehydrogenase – tetramer (four subunits)
composed of two types of polypeptide chains, M and H
There are 5 Isozymes of LDG:
 H4 – heart
 HM3
 H2M2
 H3M
 M4 – liver, muscle
• H4: highest affinity; best in aerobic environment
•M4: lowest affinity; best in anaerobic environment
Isoenzymes are important for diagnosis of different
diseases
Activation by proteolytic cleavage
• Many enzymes are synthesized as inactive precursors
(zymogens) that are activated by proteolytic cleavage
• Proteolytic activation only occurs once in the life of an
enzyme molecule
Examples of specific proteolysis
•Digestive enzymes
–Synthesized as zymogens in stomach and pancreas
•Blood clotting enzymes
–Cascade of proteolytic activations
•Protein hormones
–Proinsulin to insulin by removal of a peptide