The Biochemical Role of Enzymes
Introduction
9/10/2012
Prof. Dr. Hedef D. El-Yassin(Ph.D.,Post Doctorate)
Enzyme-Catalyzed Reactions
Enzymes are catalysts that increase the rate or velocity, v, of many physiologic reactions.In the absence of enzymes, most reactions in the body would proceed so slowly that life would be impossible.
Enzymes can couple reactions that would not occur spontaneously to an energy-releasing reaction, such as ATP hydrolysis, that makes the overall re action favorable.
Another of the most important properties of enzymes as catalysts is that they are not changed during the reactions they catalyze, which allows a single en zyme to catalyze a reaction many times.
Enzymes specifically bind the reactants in order to catalyze biologic reactions.
During the reaction, the reactants or substrates are acted on by the enzyme to yield the products.
Each substrate binds at its binding site on the enzyme, which may contain, be near to, or be the same as the active site harboring the amino acid side chains that participate directly in the reaction.
Enzymes exhibit selectivity or specificity, a preference for catalyzing reac tions with substrates having structures that interact properly with the cat alytic residues of the active site.
A deficiency in enzyme activity can cause disease.
Inherited absence or mutations in enzymes involved in critical metabolic pathwayseg, the urea cycle or glycogen metabolismare referred to as in born errors of metabolism. If not detected soon after birth, these conditions can lead to serious metabolic derangements in infants and even death.
An enzyme deficiency can produce a deficiency of the product of the reac tion it catalyzes, which may inhibit other reactions that depend on availabil ity of that product.
Accumulation of the substrate or metabolic byproducts of the substrate due to an enzyme deficiency can have profound physiologic consequences.
Most inborn errors of metabolism manifest after birth because the exchange of metabolites between mother and fetus provides for fetal metabolic needs in utero.
Therapeutic strategies for enzyme deficiency diseases include dietary modifica tion and potential gene therapy or direct enzyme replacement (Table).
Table: Example of enzyme replacement therapy for inherited disease
Physiologic Consequences and Prognosis
Major Symptoms
Or
Findings on Examination
Normal Function
of the Enzyme
Enzyme
deficiency
DiseaseGlycogen accumula tion in several organs, including heart and skeletal muscle Congestive heart failure
Weakness,
fatigue,
failure to thrive,
lethargy
Hydrolysis of
glycogenAcid α-1,4-
gluco-
sidase
Pompe disease
Accumulation of gluco-cerebroside in several organs, reduced lung and brain function, pain in upper trunk region, seizures, convulsionsEasy bruising,
fatigue,
anemia,
reduced platelet count
Hydrolysis of the glycollipid, gluco-cerebroside,
a product of
de gradation of
RBCs and WBCs
Gluco-
Cerebro-
sidase
Gaucher disease
Accumulation of globotriaosylcer-amide in endothelial cells of the blood vessels, altered cellular structure of heart and glomeruli, renal failure
Severe fatigue, painful paresthesias (numb ness and tingling) of the feet and arms, purplish skin lesions on abdomen and buttocksHydrolysis of the
lipid, globotri
aosylceramide
α-Galacto-sidase A
Fabry
diseaseEnzyme Classification
Enzymes can be made of either protein or RNA.Most enzymes are proteins, which are grouped according to the six types of reactions they catalyze..
The top-level classification is
ClassNameTrivial Names and examplesType of reaction catalyzed1OxidoreductasesDehydrogenases
Reductases
OxidasesAddition or subtraction of electrons2TransferasesKinases-Phosphotransferases
AminotransferasesTransfer of small groups: amino, acyl, phosphoryl,
one-carbon, sugar3HydrolasesGlycosidases
Nucleases PeptidasesAdd water across bonds to cleave them4LyasesDecarboxylase
Dehydratases
HydratasesAdd the elements of water, ammonia, or carbon dioxide across a double bond (or the reverse reaction)5IsomerasesMutases
EpimerasesStructural rearrangements6LigasesSynthases
SynthetasesJoin molecules together
Several important physiologic catalysts are made of RNA, and these RNA-based enzymes or ribozymes are of two general types:
RNA molecules that undergo self-splicing, in which an internal portion of the RNA molecule is removed while the parts on either side of this intron are reconnected.
Other RNA molecules that do not undergo self-splicing can act on other molecules as substrates are true catalysts.
Ribonuclease P cleaves transfer RNA precursors to their mature forms.
The 23S ribosomal RNA is responsible for the peptidyl transferase activ ity of the bacterial ribosome.
Isozymes are protein-based enzymes that catalyze the same reaction but differ in amino acid composition.
Because of their structural differences, isozymes may often be distinguished by separation in an electric field (electrophoresis) or by reactivity with selec tive antibodies.
Several clinical uses have been made of isozymes selectively expressed by dif ferent tissues.
Example:
Diagnosis of Heart Attack and Muscle Damage
The enzyme creating kinase (CK) is formed of two subunits that can either be of the brain (B) type or the muscle (M) type, and different combinations of these types lead to isozymes that predominate in the brain (BB), skeletal muscle (MM), and heart muscle (MB).
Within 3-4 hours of a heart attack, damaged myocardial cells release CK of the MB type, which can be detected in serum by a monoclonal antibody and is useful to confirm the diagnosis.
Skeletal muscle myopathy often leads to release of CK of the MM type. Rhabdomyolysis is one of the major side effects of treatment with the cholesterol-lowering drugs the statins.
Inflammation of the muscle (myositis) leads to cell death.
The condition is characterized by muscle pain, weakness, elevated CK MM, and myoglobinuria.
Mechanisms of Enzyme Catalysis
Enzymes use a variety of strategies to catalyze reactions, and individual enzymes often use more than one strategy.
Substrate binding by an enzyme helps catalyze the reaction by bringing the reac tants into proximity with the optimal orientation for reaction.
Amino acid side chains within active sites of many enzymes assist in catalysis by acting as acids or bases in reaction with the substrate.
In the mechanism of the pancreatic hydrolase ribonuclease, a specialized histidine within the active site acts as a general acid or proton donor to begin cleavage of the phosphodiester linkage of the substrate RNA.
The digestive enzyme chymotrypsin has a serine in its active site that acts as a general base or proton acceptor during hydrolysis of peptide bonds in protein substrates (Figure).
Figure from (Nelson & Cox, Lehninger Principles of Biochemistry, 3rd ed., 2000):
The binding of polysaccharide substrates that have six or more sugar groups to lysozyme, the enzyme in tears and saliva that cleaves such molecules, induces strain in the sugar nearest the active site making the nearby bond more suscepti ble to hydrolysis.
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Figure from Garrett & Grisham, Biochemistry, 2nd ed., 1999) Lysozyme mechanism
In covalent catalysis, the enzyme becomes covalently coupled to the substrate as an intermediate in the reaction mechanism before release of the products.
The active site serine of chymotrypsin attacks the protein substrate, which is cleaved and a portion of it becomes temporarily connected through the serine by an acyl linkage to the enzyme.
The acyl-enzyme intermediate reacts further by transfer of the polypeptide segment to water, completing cleavage (or hydrolysis) of the protein substrate.
Examples:
Snake Venom Enzymes: Hydrolases that Produce Toxic Effects
Snake venoms are composed of a toxic mixture of enzymes that can kill or immobilize prey.
Neurotoxic venoms of cobras, mambas, and coral snakes inhibit the enzyme acetylcholinesterase.
This hydrolase normally breaks down the neurotransmitter acetylcholine within nerve synapses.
The resultant elevation of acetylcholine causes a transient period of contraction followed by pro longed depolarization in the postsynaptic muscle cell, which induces relaxation and then paralysis of the victim.
Hemotoxic venoms of rattlesnakes and cottonmouths contain as their principal toxin phosphodiesterase, an enzyme that catalyzes hydrolysis ofphosphodiester bonds in A TP and other substrates.
One consequence of this activity is altered metabolism of endothelial cells, which leads to cardiac ef fects and rapid decrease in blood pressure.
These venoms induce circulatory shock and potentially death.
Enzymes as Therapeutic Agents
The catalytic efficiency and exquisite specificity of enzymes have been exploited for use as therapeutic agents in certain diseases.
Patients with cystic fibrosis use aerosol inhaler sprays of the DNA-hydrolyzing enzyme deoxyribonuclease to help reduce the viscosity of mucous secretions, which contain large amounts of DNA arising from destruction of WBCs as they fight lung infections.
Patients who have had a heart attack or stroke are frequently treated by intravenous administration of tissue plasminogen activator (tPA) or streptokinase, enzymes that break down fibrin clots that clog blood vessels.
Allosteric Regulation of Enzymes
Key enzymes that catalyze rate-limiting steps of metabolic pathways or that are responsible for major cellular processes must be regulated to maintain homeostasis of individual cells and the organism overall.
Allosteric regulation refers to binding of a molecule to a site on the enzyme other than the active site and induces a subsequent change in shape of the enzyme causing an increase or decrease in its activity.
Many allosteric enzymes have multiple subunits whose interaction accounts for their unusual kinetic properties.
Enzymes that are subject to allosteric regulation by either positive or negative effectors exhibit cooperativity.
2. In the presence of positive cooperativity, a plot of v versus [S] shows sigmoidal kinetics, ie, is S-shaped (Figure).
This kinetic behavior signifies that the enzyme's affinity for the substrate increases as a function of substrate loading.
This is analogous to O2 binding by hemoglobin, in which O2 loading to one subunit facilitates O2 binding to the next subunit, and so on.
Figure: Relationship between V; and [S] for a reaction catalyzed by an allosteric enzyme, showing the effects of positive and negative effectors.
Feedback inhibition occurs when the end product of a metabolic pathway accumulates, binds to and inhibits a critical enzyme upstream in the pathway, ei ther as a competitive inhibitor or an allosteric effector.