Download Structure and physical-chemical properties of enzymes

Structure and physical-chemical properties of enzymes.
Enzymes - catalysts of biological reactions
Accelerate reactions by a millions fold
Common features for enzymes and
inorganic catalysts:
1. Catalyze only thermodynamically possible
reactions
2. Are not used or changed during the reaction.
3. Don’t change the position of equilibrium and
direction of the reaction
4. Usually act by forming a transient complex with
the reactant, thus stabilizing the transition state
Specific features of enzymes:
1. Accelerate reactions in much
higher degree than inorganic
catalysts
2. Specificity of action
3. Sensitivity to temperature
4. Sensitivity to pH
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
Active site of lysozym consists of six amino acid
residues which are far apart in sequence
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
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
Kinetic properties of enzymes
Study of the effect of substrate concentration on the rate of reaction
Leonor Michaelis and Maud Menten – first researchers
who explained the shape of the rate curve (1913)
During reaction enzyme molecules, E, and substrate
molecules, S, combine in a reversible step to form an
intermediate enzyme-substrate (ES) complex
E + S
k1
k-1
ES
k2
E + P
k-2
k1, k-1, k2, k-2 - rate constant - indicate the speed
or efficiency of a reaction
Rate of Catalysis
- At a fixed enzyme concentration [E],
the initial velocity Vo is almost linearly
proportional to substrate concentration
[S] when [S] is small but is nearly
independent of [S] when [S] is large
- Rate rises linearly as [S] increases and
then levels off at high [S] (saturated)
The Michaelis-Menten Equation
The basic equation derived by Michaelis and Menten to explain
enzyme-catalyzed reactions is
Vmax[S]
vo =
Km + [S]
Km - Michaelis constant;
Vo – initial velocity caused by substrate concentration,
[S];
Vmax – maximum velocity
Effect of enzyme concentration [E]
on velocity (v)
In fixed, saturating
[S], the higher the
concentration of
enzyme, the greater
the initial reaction
rate
This relationship will
hold as long as there
is enough substrate
present
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
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
Principle of the international classification
All enzymes are classified into six categories
according to the type of reaction they catalyze
Each enzyme has an official international name
ending in –ase
Each enzyme has classification number
consisting of four digits: EC: 2.3.4.2
First digit refers to a class of enzyme, second to a subclass, third – to a subsubclass, and
fourth means the ordinal number of enzyme in
subsubclass
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)
An important first step in restoring
health and well-being by helping to
remedy digestive problems. Food
(plant) enzymes and pancreatic
(animal) enzymes are used in
complementary ways to improve
digestion and absorption of essential
nutrients. Treatment includes
enzyme supplements, coupled with
healthy diet that features whole
foods. Plant-derived enzymes and
pancreatic enzymes can be used
independently or in combination.
A chart of the numerous digestive enzymes of
the body and their functions
.
• Amylasedigests starchesBromelaina proteolytic, anti-inflammatory
food enzyme from pineapple. Aids digestion of fatsCatalaseworks
with SOD to reduce free radical productionCellulasedigests cellulose,
the fibrous component of most vegtable matter Chymotrypsinhelps
convert chyme Diastasea pontent vegtable starch
digestantLactasedigests lactose, or milk sugar, (almost 65% of humans
are deficient).Lipasedigests fats.Mycozymea single-celled plant
enzyme for digestion of starches.Pancreatina broad spectrum,
proteolytic digestive aid, derived from secretions of animal pancreas;
important in degenerative disease research. Papin and
chymopapainproteolytic food enzymes from unripe papaya; a
veegatable pepsin for digesion of proteins. These enzymes help loosen
nercotic and encrusted waste material from the intestinal walls.Pepsina
proteolytic enzyme that breaks down proteins into peptides. Can digest
3500 times its weight in proteins.Proteasedigests proteinsRenninhelps
digest cow's milk products.Trypsina proteoytic enzyme
enzymopathy
• Any of various disturbances of enzyme
function, such as the genetic deficiency of a
specific enzyme.
Celiakia
INBORN ERRORS OF AMINO ACIDS METABOLISM
Alcaptonuria - inherited disorder of the
tyrosine metabolism caused by the
absence of homogentisate oxidase.
 homogentisic acid is accumulated and
excreted in the urine
 turns a black color upon exposure to air
 In children:
 urine in diaper may
darken
 In adults:
 darkening of the ear
 dark spots on the on the
sclera and cornea
 arthritis
Maple syrup urine disease - the disorder of the
oxidative decarboxylation of -ketoacids derived
from valine, isoleucine, and leucine caused by the
missing or defect of branched-chain dehydrogenase.
The levels of branched-chain amino
acids and corresponding -ketoacids
are markedly elevated in both blood
and urine.
The urine has the odor of maple syrup
The early symptoms:
 lethargy
 ketoacidosis
 unrecognized disease leads to
seizures, coma, and death
 mental and physical retardation
Phenylketonuria is caused by an absence or deficiency
of phenylalanine hydroxylase or of its
tetrahydrobiopterin cofactor.
Phenylalanine accumulates in all body fluids and converts
to phenylpyruvate.
Defect in
myelination of nerves
The brain weight is
below normal.
Mental and physical
retardations.
The life expectancy
is drastically
shortened.
Diagnostic criteria:
 phenylalanine level in
the blood
 FeCl3 test
 DNA probes
(prenatal)