Enzymes: Catalysts and Ribozymes

Enzymes: Definition

• Enzymes act as catalysts to accelerate chemical reactions.
• A catalyst is a substance that heightens the rate of a chemical reaction.

Nature of Enzymes

• Most enzymes are proteins, except for a few catalytic RNA molecules known as ribozymes.
• Enzymes can either be simple or conjugated proteins.
• Enzymes share the same properties as proteins, including denaturation, precipitation, and electrophoresis.

Ribozymes

• Ribozymes are ribonucleic acid molecules that catalyze reactions on the phosphodiester bond of other RNAs.

Localization of Enzymes

• Enzymes are either intracellular (in mitochondria, cytoplasm, or both) or extracellular (e.g., digestive enzymes like pepsin and trypsin).
• Enzymes exist in all tissues and fluids of the body.
• Plasma membrane enzymes regulate catalysis within cells in response to extracellular signals.
• Enzymes within the circulatory system are responsible for regulating blood clotting.
• Nearly every significant life process depends on enzyme activity.

Enzyme Deficiencies

• Deficiencies in the quantity or catalytic activity of key enzymes can result from genetic defects or mutations, nutritional deficits, exposure to toxins, or infections caused by viruses or bacteria like Vibrio cholerae.

Common Features of Enzymes

• Produced by living cells.
• Needed in very small amounts.
• Accelerate reactions without affecting equilibrium.
• Remain chemically unchanged at the end of a reaction.
• Highly specific.

Mechanism of Action: Catalysis at Active Site

• Each enzyme has a specific catalytic site where substrates bind to form an enzyme-substrate complex, facilitating rapid chemical reactions.
• The catalytic site is in the form of a cleft or pocket.
• The active site also binds cofactors or prosthetic groups.

Allosteric Site

• Another active site may be present on the enzyme surface, far from the catalytic site.
• It can combine with small molecules called allosteric effectors.
• This combination can either inhibit or increase enzyme activity.

Substrate Definition

• A substrate is a substance involved in a reaction catalyzed by an enzyme.
• Each enzyme has its own specific substrate.

Enzyme-Substrate Complex Models: Lock and Key Model

• Lock and key theory, also known as the Fischer model proposes that the active site of an enzyme is complementary in conformation to its substrate, allowing them to "recognize" each other.
• There is a highly specific fit between the enzyme and its substrate, so no conformational change occurs in the enzyme.

Enzyme-Substrate Complex Models: Induced-Fit Model

• The Koshland theory states that the binding of a substrate to an enzyme induces a conformational change, similar to fitting a hand (substrate) into a glove (enzyme).
• This model posits that the enzyme changes shape as the substrate binds.

Mechanisms of Catalysis: Catalysis by Proximity

• Molecules must come within bond-forming distance of each other to react.

Mechanisms of Catalysis: Acid-Base Catalysis

• Ionizable functional groups of prosthetic groups (metals) help in catalysis by acting as acids or bases.

Mechanisms of Catalysis: Catalysis by Strain

• Enzymes catalyze the breaking of a covalent bond, by binding substrates in a slightly unfavorable conformation for the bond that needs breaking.
• The resulting strain stretches or distorts the targeted bond, making it easier to break.

Mechanisms of Catalysis: Covalent Catalysis

• The process involves forming a covalent bond between an enzyme and one or more substrates.
• The modified enzyme becomes a reactant, help the reaction to be faster.
• Residues of the enzyme participating in covalent catalysis are cysteine (SH) and serine (OH).
• Covalent catalysis often follows a "ping-pong" mechanism, where the first substrate is bound and its product released before binding the second substrate.

Definition of Isozymes

• Isozymes (or iso-enzymes) are variant forms of enzymes with the same catalytic action but differences in chemical structure.
• They originate from different organs and arise through gene duplication.
• Isozymes can be distinguished via electrophoresis, centrifugation, or immunological methods.
• They aid in medical diagnosis.

Examples of Isozymes

• Lactate dehydrogenase (LDH).
• Creatine phosphokinase (CK).

Lactate Dehydrogenase (LDH)

• LDH is a tetramer with four subunits occurring in two isoforms: H (heart) and M (muscle).
• The subunits combine to form five isozymes: LDH I1 (HHHH), LDH I2 (HHHM), LDH I3 (HHMM), LDH I4 (HMMM), and LDH I5 (MMMM).
• LDH I1 is more prevalent in the heart.
• LDH I1 levels increase markedly in plasma during myocardial infarction (3-10 hours after occurrence).
• LDH I5 is more prevalent in the liver and skeletal muscle.
• LDH I5 levels increase in cases of hepatitis and muscle diseases.
• These enzymes are thus important for clinical diagnosis.

Creatine Kinase (CK)

• Creatine Kinase is a dimer formed of two subunits M and B.
• There are three isozymes: CK1 (CK-BB present in the brain, elevated in brain infarction), CK2 (CK-MB present in the heart, elevated 2-3 hours after myocardial infarction), and CK3 (CK-MM present in skeletal muscle, elevated in muscle diseases like trauma).

Clinical Importance of Plasma Enzymes

• Plasma enzymes are classified as functional or non-functional.

Functional Plasma Enzymes

• Substrates are always present in the circulation of normal individuals and perform physiological functions in the blood.
• Most are synthesized in and secreted by the liver (e.g., lipoprotein lipase, pseudocholine esterase, and proenzymes of blood coagulation).

Non-Functional Plasma Enzymes

• They do not serve a function in the blood.
• They are synthesized in their target organs.
• Normally present in blood at low concentrations.
• Tissue damage or necrosis (e.g., hepatitis, myocardial infarction) due to injury or disease leads to the release of these enzymes into the circulation, markedly increasing their blood levels, making them useful for clinical diagnosis.

Enzymes Used in Medical Diagnosis

• Liver:
  • Serum enzymes: Aminotransferase [Alanine aminotransferase (ALT or SGPT), Aspartate aminotransferase (AST or SGOT)], Ceruloplasmin, y-Glutamyl transpeptidase (GGT) or (γGT), Lactate dehydrogenase (LDH I5) (LDH MMMM), Alkaline phosphatase.
  • Diagnosis: Viral Hepatitis, Hepatolenticular degeneration "Wilson diseases", Various liver disease, Hepatitis, Obstructive liver disease (obstructive jaundice).
• Bone:
  • Serum enzymes: Alkaline phosphatase
  • Diagnosis: Bone disorders "rickets"
• Pancreas:
  • Serum enzymes: Amylase, Lipase
  • Diagnosis: Acute pancreatitis
• Prostate:
  • Serum enzymes: Acid phosphatase, Prostatic specific antigen (PSA).
  • Diagnosis: Carcinoma of the prostate
• Brain:
  • Serum enzymes: Creatine Kinase (CK BB) or (CK1)
  • Diagnosis: Brain infarction
• Muscle:
  • Serum enzymes: Creatine Kinase (CK MM) or (CK3), Lactate dehydrogenase (LDH I5) (LDH MMMM)
  • Diagnosis: Muscle diseases e.g. trauma
• Heart:
  • Serum enzymes: Troponin I (cTnI) or T (cTnT), Creatine Kinase (CK MB) or (CK2), Aminotransferase, Aspartate aminotransferase(AST or SGOT), Lactate dehydrogenase (LDH I₁) (LDH HHHH)
  • Diagnosis: Myocardial infarction

Cardiac Enzymes

• Several enzymes are released when heart cells are damaged.
• These serve as specific, sensitive markers.

Troponin T and I

• These are contractile proteins of the myofibril.
• The cardiac isoforms are very specific for cardiac injury (myocardial infarction) and are absent in the serum of healthy people.
• The American College of Cardiology states that cardiac troponins are the preferred markers for detecting myocardial cell injury.
• Troponin I (cTnI) or T (cTnT) are the forms frequently assessed.
  • Rise 2-6 hours after injury.
  • Reach peak in 12-16 hours.
  • cTnI stays elevated for 5-10 days, cTnT for 5-14 days.

Creatine Kinase (Creatine Phospho-Kinase) CK-MB

• The enzyme is found in the heart muscle (CK-MB) isoform.
  • Elevated in over 90% of myocardial infarctions.
  • Begins to rise 4-6 hours.
  • Peaks 24 hours after occurrence.
  • Returns to normal in 3-4 days.

Aspartate Amino Transferase (AST)

• Also known as (GOT).

Lactate Dehydrogenase (LDH I1)

• Also known as (LDH HHHH).
• Aspartate Amino Transferase (AST) or (GOT).

Diagnostics and Cardiac Enzymes

• AST and LDH enzymes are no longer used to diagnose myocardial infarction.

Enzymes Facilitate Diagnosis of Genetic Diseases

• Polymerase Chain Reaction (PCR) is a technique for DNA amplification using a thermostable DNA polymerase and specific primers to produce thousands of copies of a DNA sample.
• The technique helps diagnose important diseases like hepatic virus C infection (HCV).
• Restriction endonucleases aid in the prenatal detection of hereditary disorders such as sickle cell trait and beta-thalassemia.

Important Therapeutic Enzymes: Streptokinase

• Produced by streptococci.
• It is an exogenous activator of plasminogen; (plasminogen → plasmin).
• Used to dissolve thrombi (a thrombolytic agent) in conditions like myocardial infarction.

Important Therapeutic Enzymes: Urokinase

• Activates plasminogen to plasmin, used to dissolve blood clots.

Important Therapeutic Enzymes: Hyaluronidase

• Causes hydrolysis of hyaluronic acid and is used in the treatment of heart attacks.

Important Therapeutic Enzymes: Asparaginase

• Causes hydrolysis of asparagine to aspartate.
• Used in the treatment of leukemia.

Important Therapeutic Enzymes: Glutaminase

• Used in the treatment of leukemia.

Factors Affecting Enzyme Action & Kinetics: pH

• Changes in pH can affect the rates of enzyme-catalyzed reactions.
• Every enzyme has an optimum pH at which it functions at a maximum rate.
• The optimum pH for pepsin is 1-2, for chymotrypsin is 8.0, and for salivary amylase is 6.8.
• Strong acids and alkalis cause denaturation of enzymes.

Factors Affecting Enzyme Action & Kinetics: Temperature

• Most enzymes are thermolabile.
• The rate of an enzyme-catalyzed reaction increases with increasing temperature up to an optimum point, the optimum temperature.
• An increase in temperature beyond the optimum point leads to a decrease in enzymatic activity.
• Enzymes in the human body are homeothermic and tolerate temperatures from 37-40°C.
• Freezing can preserve enzyme activity.

Factors Affecting Enzyme Action & Kinetics: Enzyme Concentration

• The rate of the enzyme-catalyzed reaction increases with increasing enzyme concentration, until a certain point.

Factors Affecting Enzyme Action & Kinetics: Cofactors, Coenzymes, and Prosthetic Groups

• The reaction rate increases with the increase of the factors needed for enzyme action.

Factors Affecting Enzyme Action & Kinetics: Irradiation

• UV and X-ray: All cause enzyme inactivation and denaturation.

Factors Affecting Enzyme Action & Kinetics: Product Inhibition

• In some enzymatic reactions, the product can inhibit the enzyme if it accumulates, limiting the rate of product formation.

Effect of Substrate Concentration on Reaction Velocity

• Increasing the substrate concentration [S] will increase the reaction velocity [Vi] until it reaches a maximum value (Vmax).
• When the enzyme is saturated, further increases in substrate concentration do not increase Vi.

Michaelis-Menten Equation

• The Michaelis-Menten equation illustrates the relationship between initial reaction velocity [Vi] and substrate concentration [S]:Vi = (Vmax [S]) / (Km+ [S]).
• The Michaelis constant (Km) is the substrate concentration at which Vi is half the maximal velocity (Vmax/2) and the enzyme is half saturated with its substrate.
• Smaller Km values reflect higher enzyme affinity for its substrate, and larger Km values reflect lower affinity.

Linear Transforms (Lineweaver-Burk)

• The Lineweaver-Burk plot transforms the hyperbolic curve to a linear one, and is more useful to estimate Vmax and determine the Km value.

Enzyme Activation: Zymogens

• A zymogen is an inactive enzyme where the catalytic site is covered by a peptide.
• Some enzymes are secreted in zymogen form; the best examples are digestive enzymes.

Enzyme Activation: Auto Activation

• Auto-activation occurs when the enzyme activates its zymogen form:
  • Pepsinogen → pepsin.
  • Trypsinogen → trypsin.

Enzyme Activation: Activation by HCl

• Occurs "by hydrogen ions"
  • Pepsinogen + (H+) → Pepsin

Enzyme Activation: Pancreatic Enzymes

• Pancreatic enzymes by specific intestinal enzyme:
  • Trypsinogen + Enterokinase → trypsin

Enzyme Activation: Blood Clotting Factors

• Blood clotting factors are formed as zymogens by some proteases.

Specific Metal Ion Activation

• Certain metal ions activate specific enzymes:
  • Mg++ activates kinases.
  • Fe++ activates catalase.
  • Zn++ activates carbonic anhydrase.
  • Ca+ activates lipase.
  • Mn++ activates carboxylases.

Allosteric Activation

• Some small molecules bind to the allosteric site of enzymes.
• This site is usually separate to the catalytic site of the enzyme itself
• This leads to enzyme activation and increased reaction velocity.
• Called positive allosteric modifiers or activators.

Covalent Modification

• Phosphorylation-Dephosphorylation Mechanism.
• Many enzymes are activated by this mechanism.
• The active form may be phosphorylated or dephosphorylated.
• Phosphorylation involves adding a phosphate group via a covalent bond.
• This requires "protein kinases enzymes" in the presence of ATP, adding a phosphate group to the OH group of serine, threonine, or tyrosine.
• Dephosphorylation involves removing a phosphate group.
• It is helped by specific phosphatases enzymes, which cleave the covalent bond.

Competitive Inhibition

• Occurs when the substrate [S] and an inhibitor [I] compete for binding to the enzyme's active site.
• The inhibitor and substrate are structurally similar (substrate analogs).
• This inhibition is reversible by increasing the substrate concentration.
• Competitive inhibition increases the Km of the enzyme but has no effect on the Vmax.
• The inhibitor forms an enzyme-inhibitor complex [EI] equivalent to [ES].

Non-Competitive Reversible Inhibition

• The inhibitor binds at a site on the enzyme, but the substrate binding is unaffected as the inhibitor binds at the allosteric site of the enzyme.
• The binding of the inhibitor blocks catalytic activity, decreasing the amount of active enzyme and reducing Vmax but not affecting Km.
• This inhibition can be reversed, but not by increasing the substrate concentration.
• e.g., the inhibition of Mg2+, a cofactor of enolase, can be reversed by adding more Mg2+.

Non-Competitive Irreversible Inhibition

• The inhibitor binds irreversibly to the enzyme, removing the enzyme from the reaction, via binding to an allosteric site.
• The Vmax is decreased, but the Km is unchanged.
• Examples included denaturing agents such as, strong acids, strong alkalis, UV rays, and high temperature.
• Oxidizing agents: block the active free SH group of enzymes to form a di-sulfide bond.
• Gluthathione can protect intracellular enzymes from oxidation
• Iodoacetate: will form an alkyal radical, inactivating the enzyme.
• Heavy metals as PB, Cu and Hg form.

Allosteric Inhibitors (Feedback Inhibitors)

• Feedback inhibition is inhibiting an enzyme in a biosynthetic pathway by the end product.
• High concentrations of the end product (D) inhibit the conversion of A to B by binding allosterically to and inactivating enzyme 1.
• Feedback inhibitors have little/no structural similarity to the substrates of the enzymes they inhibit and act as an allosteric effector.

Regulation of Enzyme Activity

• Through metabolic pathways (synthetic and catabolic), the enzymes of each pathway are regulated to achieve required body requirements.

Regulation of Enzyme Activity: Covalent Modification

• "phosphorylation -Dephosphorylation mechanism": The active form may be phosphorylated or dephosphorylated through protein kinases enzymes Phosphorylation:
• Helps by specific phosphatases enzyme, which cleave the covalent bond.

Regulation of Enzyme Activity: Allosteric Modification

• Activation occurs by a positive allosteric modifier "allosteric activators"; while inhibition is achieved by negative allosteric modifiers "allosteric inhibitors".

Regulation of Enzyme Activity: Activation of Zymogens

• Activation of zymogens. A zymogen is an inactive enzyme where there catalytic site is covered by a peptide.
• Digestive enzymes are secreted in the digestive tract as zymogens.
• An example is auto-activation: pepsinogen is activated to pepsin.
• Trypsinogens are activated to trypsin.

Regulation of Enzyme Activity:

• Blood clotting factors activate as zymogens by proteases.

Regulation of Enzyme Activity: Hormonal Regulation

• Hormones stimulate protein kinases by cAMP.
• Some hormones stimulate the synthesis of protein synthesis at the ribosomal level e.g. steroid hormones.

Enzyme Specificity

• Absolute specificity: catalyze only one particular reaction.
• Dual specificity: catalyze two particular reactions.
• Group specificity: act only on molecules with specific functional groups.
• Linkage specificity: act on a particular type of chemical bond.
• Stereochemical specificity: act on a particular stereoisomer or optical isomer.

Enzyme Classification

• There are 6 classes of enzymes are classified according to the International Union of Biochemistry (IUB)
  • Oxidoreductases, Transferases, Hydrolases, Lyases, Isomerases, Ligases

Vitamins: Definition

• Vitamins are organic compounds that cannot be synthesized by human tissues but are required for normal growth and development.
• Vitamins have to be in a the diet as only small molecules are required to treat vitamin deficiency.

Vitamins: Sources

• Humans have two sources of vitamins: through food (all vitamins), or through intestinal microorganisms.

Vitamins Deficiency: Causes

• Inadequate intake. Inadequate absorption resulting from biliary obstruction, or diseases of the intestine.
• Inadequate use due to failure to synthesize the active form of vitamins, or increased excretion.

Drugs Induced Vitamins Deficiency

• Antibiotics treatment leads to a loss microorganism synthesis.
• Pyridoxine (B6) deficiency leads patients receiving Isoniazid for the treatment of tuberculosis.

Vitamins: Classification

• Fat soluble vitamins are the soluble in non polar solvent ( Vitamins A, D, K, E) while soluble in water (Vitamin C and B complex group
• Fat soluble need bile salt and lipids during absorption, whereas water absorption is simple.
• There are single large doses for fat soluble, whereas dietary supply is required for water soluble. Fat soluble:
  • Major Vitamins -A,D, K, E
  • Solubility in fat - soluble
  • Absorption - requires the present of lipid and bile salt
  • Storage - stored in liver
  • Excretion - Not excreted
  • Toxicity - Hyper can occur
  • Treatment of Single: large does may prevent deficiency

Water Soluble: - - Major Vitamins -C &B complex group - Solubility in water - soluble - Absorption -Simple, except Vit.B12 - Carrier protein - Not required, except Vit.B12 - Storage -Not storage, except Vit.B12 - Excretion-Excreted, except Vit.B12 - Deficiency -Manifests rapidly as there is no storage except Vit.B12 - Toxicity - Unlikely; since excess is excreted in urine

Fat Soluble Vitamins

• Retinoid:
  • Role vision or diff of immune cells.
  • Source leafy greens/vegetables and fish oils.
  • Early sign is the loss of sensitivity to sunlight.

Vitamin D:

• Source is egg yolk, is a steroid hormone.
• Role is to maintain plasma level with intestine or mineral support.

Steryl antioxidant

Source from green veggies, or seed oils to protect nerves. The lack of vitamins may cause neurological dysfunction, with extreme deficiency.