Enzymes and Biochemistry

• This section consists of 12 lectures covering enzymes, vitamins, hormones, minerals, amino acid metabolism, and the urea cycle.
• The class will use "Lippincott's Illustrated Reviews: Biochemistry 6th Edition," with copies in English and turkish.
• Required Texts for lab are “Experimental Biochemistry for Medical Sciences Students” by T. Yigitbasi, S.S.Erdem, P.Bozaykut, and “Lehningers Principals of Biochemistry”.

Learning Objectives:

• Cover:
  • Major enzyme classes
  • Enzyme properties
  • Enzyme kinetics
  • Enzyme inhibition
  • Mechanisms regulating enzyme activity
  • Enzymes in clinical diagnosis

Overview of Enzymes:

• Enzymes are biological catalysts.
• Enzymes speed up biochemical reactions.
• Most are globular 3D proteins.
• Enzymes have tertiary and quaternary structures.
• Enzymes direct metabolic events by selectively channeling substrates into pathways.

Rate Enhancement

• The rate enhancement by enzymes can be significant, ranging from 10^6 to 10^17 times faster compared to non-enzymatic reactions.

Enzyme Nomenclature:

• Recommended name:
  • Short and for everyday use.
  • The suffix "-ase" is added to the substrate or action description. (e.g., glucosidase, lactate dehydrogenase).
• Systematic name:
  • It is a more complex classification developed by the International Enzyme Commission.
  • Enzymes are divided into six major classes with numerous subgroups, each with the "-ase" suffix.

Major Enzyme Classes

• Oxidoreductases:
  • Catalyze oxidation-reduction reactions.
• Transferases:
  • Transfer C-, N-, or P-containing groups.
• Hydrolases:
  • Cleave bonds by adding water.
• Lyases:
  • Cleave C-C, C-S, and certain C-N bonds.
• Isomerases:
  • Catalyze racemization of optical or geometric isomers.
• Ligases:
  • Form bonds between carbon and O, S & N, coupled to hydrolysis of high-energy phosphates.

Enzyme Synthesis Sites

• Enzymes are synthesized by ribosomes attached to rough endoplasmic reticulum.
• DNA carries information for enzyme synthesis.
• Specific enzymes are formed as amino acids bond together based on DNA codes.

Intracellular and Extracellular Enzymes

• Intracellular Enzymes:
  • Synthesized and retained for cellular use
  • Found primarily in the cytoplasm, nucleus, mitochondria & chloroplasts.
  • Oxydoreductase catalyzes biological oxidation.
  • Enzymes participate in mitochondrial reduction.
• Extracellular Enzymes:
  • Synthesized inside cells but secreted for external work.
  • Digestive pancreatic enzymes are transported to the duodenum.

Enzyme Properties:

• Enzymes are protein catalysts that increase reaction velocity.
• They are highly specific, acting only on one substrate.
• Enzymes have absolute specificity.
• Enzymes are localized in cell compartments.
• Stereospecificity:
  • The ability to differentiate between optical isomers.
• Reaction Specificity:
  • Enzymes that catalyze certain types of reactions.

Active Site

• The active site of an enzyme is a small pocket or cleft.
• It constitutes less than 5% of the enzyme's total surface area.
• Active sites have amino acid side chains for substrate binding.
• Binding induces conformational changes, enabling catalysis.

Enzyme Efficiency

• Enzyme-catalyzed reactions are highly efficient.
• Reactions proceed 10^3 to 10^8 times faster.
• Turnover Number (kcat):
  • The number of substrate molecules converted to product per enzyme molecule per second.
  • kcat typically ranges from 10^2 to 10^4 s^-1.

Holoenzymes, Apoenzymes, Cofactors, and Coenzymes:

• Some enzymatic activity requires non-protein molecules.
• Holoenzyme:
  • Is an active enzyme with its nonprotein component.
• Apoenzyme:
  • Is an inactive enzyme without its nonprotein component.
• Nonprotein Part:
  • Coenzymes, cofactors, etc.
• Prosthetic Group:
  • Permanently associated coenzyme (e.g., FAD).
• Apoenzyme (inactive) + Coenzyme (active) = Holoenzyme (active

Non-Protein Parts:

• Cofactors are non-protein molecules that carry out chemical reactions that standard amino acids cannot.

Types of cofactors

• Organic cofactors (coenzymes).
• Inorganic cofactors.

Inorganic Cofactors are inorganic molecules:

• Are required for proper enzyme activity.
• Carbonic anhydrase requires Zn.
• Hexokinase needs Mg.

Coenzymes (Organic Co-factors):

• Derived from vitamins and required for enzyme activity.
• E.g., Glycogen phosphorylase needing pyridoxal phosphate.
• NAD comes from niacin.
• FAD from riboflavin.

Types of Organic Co-factors:

• Prosthetic Group: Example: Flavins, heme groups, and biotin
  • Tightly bound organic co-factor.
• Coenzyme: Example: NAD+
  • Loosely bound organic co-factor.

Types of Co-factors

• An enzyme without its co-factor is called an apoenzyme.
• A protein with necessary small molecules (metal ions, other components) is a holoenzyme.

How Enzymes Work

• Enzyme catalytic efficiency is explained through:
  • Thermodynamic changes
  • Processes at the active site

Thermodynamic Changes:

• Enzymes increase reaction rates.
• They provide an alternate pathway for conversion of substrate into products.
• Only a few substances can cross the activation barrier.
• Consequently, uncatalyzed reactions proceed slowly.
• Increased reaction rate: Enzymes increase the reaction rate many folds.
• System Energy: The system's total energy remains constant.

Lock and Key Model:

• Proposed by Emil Fischer in 1894.
• Assumes active site shape is rigid.
• There is no change in the active site before and after the chemcial reaction.

Induced Fit Model:

• Proposed by Daniel Koshland in 1958.
• Enzyme exposure to substrate causes a change in the enzyme.
• It allows active site to change its shape to allow enzyme-substrate binding.

Active Site Chemistry:

• Transition state stabilization increases the concentration of the reactive intermediate.
• Active site catalytic groups enhance transition state probability.
• Analogy:
  • Baby = substrate
  • Parent = enzyme
  • Undressed baby = product

Introduction to Enzyme Kinetics:

• Enzyme kinetics is the study of enzyme catalyzed reaction rates.
• Kinetic analysis reveals the number and order of individual steps as enzymes transform substrate into product.
• An enzyme's kinetics can reveal its catalytic mechanism and how its activity is regulated.

Factors Affecting Reaction Velocity:

• Substrate Concentration:
  • Reaction rate (v) increases with substrate concentration.
  • [Substrate] correlates to velocity until max. velocity.
  • Most show Michaelis-Menten kinetics with hyperbolic shape.
  • Allosteric enzymes disobey Michaelis-Menten kinetics and have a sigmoidal curve.

Other Factors Affecting Reaction Velocity:

• Temperature:
  • Enzymes for humans can experience stability temperature increase up to 35 - 45 °C.
  • An optimal temperature helps enzymatic reactions.
• pH:
  • Proton concentrations affect reaction velocity as catalytic activity requires ionized or unionized forms.
  • Extreme pHs may denature enzymes.
  • Most enzymes have optimal activity in pH 5-9.

Michaelis-Menten Kinetics:

• The Model:
• An enzyme reversibly combines with a substrate to form an ES complex.
• This yields product and regenerates free enzyme.

Michaelis-Menten Equation:

• Describes how reaction velocity varies with substrate concentration.
• Vo = (Vmax[S]) / (Km + [S])
• Vo is the initial reaction velocity.
• Vmax is the maximum velocity.
• Km is the Michaelis constant = (k-1 + k2) / k1.
• [S] is the substrate concentration.

Assumptions for Michaelis-Menten Equation:

• Relative concentrations of enzyme E and substrate S.
  • [S] >> [E].
• The steady-state of ES does not change with time.
  • The rate of ES formation is equal to the rate of breakdown.
• The rate of the reaction is measured as soon as enzyme and substrate are mixed. The back-rxn of product to substrate is negligible.

Conclusions

• Km represents an enzyme's affinity for its substrate.
• The Michaelis-Menten constant is characteristic of an enzyme.
• Km = [S] at ½ Vmax.
• Km is inversely proportional to affinity.
• If [E] is halved, Vo and Vmax are also halved.
• When [S] << Km, the velocity ~ [S], resulting in a 1st order of rxn.
• When [S] >> Km, the velocity = Vmax, representing a 0 order rxn that is independent of [S].

Lineweaver-Burk Plot:

• It's a double-reciprocal plot.
• Used to calculate Km and Vmax.
  • It's difficult to determine when Vmax is reached given the gradual upward slope of the graph.

Enzyme Activity Inhibition:

• It prevents an enzyme process as a an action brought by the interaction between inhibitors with enzyme.
• Inhibitors:
  • Any compound lowering enzyme catalyzed reaction rates.

Inhibition Types:

• Irreversible Inhibitors:
  • Bind to enzymes through covalent bonds.
• Reversible Inhibitors:
  • Bind to enzymes through non-covalent bonds.
  • Complex dilution leads to dissociation of the reversibly bound inhibitor.

Irreversible Inhibition:

• Involves the covalent attachment of an inhibitor to the enzyme.
• It causes loss of the enzyme's catalytic activity.
• Enzymatic reactions are only restored by synthesizing molecules.

Irreversible Inhibition Examples:

• Aspirin is an irreversible inhibitor.
• Covalently modifies key enzymes involved in inflammation.

Competitive Inhibition:

• Inhibitors compete with substrate for the active site.
• The Formation of E.S complex is reduced: as a new E.I complex is formed.
• The Effect of inhibitor decreases as [S] increases.
  • At a certain [S], Vmax can be reached.
  • With more substrate, the effects of inhibitors lessen.
• Inhibitor: increases Km, which means more substrate is needed to reach ½ Vmax in the prescence of inhibitor.
• Has a characteristic Lineweaver-Burk plot: shows inhibited and uninhibited reactions intersect on the y axis at ½ Vmax.

Competitive Inhibition Example:

• Statin drugs inhibit HMG-CoA reductase in the cholesterol synthesis rate limiting step.
• Analog drugs that compete effectively for this enzyme.

Noncompetitive Inhibitor:

• It does not compete for the active site.
• It bind to an allosteric site.
• It cannot be overcome by increasing [S].
• It does not interfere with substrate binding; Km does not change.
• It does lower Vmax.

Enzyme Activity Regulation:

• Cells use regulation to turn on, turn off, or modulate metabolic pathways by regulating enzyme activity output.
• A great demand for a product may spark pathways to produce more necessary substances.
• [S] ~ reaction velocity.
• Enzyme activity is altered through allosteric effectors and/or covalent modification.

Allosteric Enzymes Regulation:

• Allosteric Enzymes:
  • Change conformation upon effector binding.
  • This causes a change in apparent binding affinity at a different ligand binding site.
• Effectors:
  • Regulate allosteric enzymes by binding noncovalently at alternate sites.
• Positive and Negative modifiers:
  • They Affect enzymatic activity.
  • These have the capability to affect affinity (Kd), Vmax or both.

Homotropic Effectors:

• This shows the substrate as the effector.
• An allosteric substrate functions as a positive effector.
• Shows sigmoidal curve pattern.
  • Contrasts enzymes with hyperbolic curves (Vo vs. [S]), following Michaelis-Menten kinetics.

Heterotropic Effectors:

• Heterotropic effectors describes a different from substrate.
• It is also a form of feedback which provides appropriate product amount by regulating substrate flow.

Regulation of Enzymes through Covalent Modification:

• Enzymes can have their activity controlled by either adding or removing phosphate groups.
• Protein Kinases:
  • Phosphorylation, often mediated by hormones, is catalyzed.
• Phosphatases:
• Enzymes which Cleave phosphate groups.

Enzyme Synthesis Induction and Repression:

• Cells have an active regulatory role through synthesis and degradation.
• The total population of active sites are altered through synthesis (induction) and degradation (repression).
• For example, elevated levels of insulin can cause increase in the synthesis of enzymes in glucose metabolism.
• Enzymes that are constant use are not regulated by rate of synthesis.

Mechanisms for Regulating Enzyme Activity:

• Substrate Availability.
• Product Inhibition.
• Allosteric Control.
• Covalent Modification.
• Synthesis or Degradation of Enzyme.

Enzymes in Clinical Diagnosis:

• Plasma enzymes split into groups:
  • Actively secreted into the blood by tissues. Liver secretion of zymogen triggers blood coagulation.
  • Many enzymes are discharged in normal cell turnover. Increased plasma levels may indicate tissue damage.

Alteration

• Alterations in plasma enzyme levels result diseases.
• Activity of many enzymes are routinely checked for in purposes of diagnosis related to diseases of key organs.

As diagnostic tools

• Some Enzymes: display high tissue activity. Increased plasma levels suggest tissue damage.
  • Liver function tests include ALT (alanine aminotransferase).
• Isoenzymes catalyze the same reactions but differ structurally and are separated by AA electrophoresis.

Diagnosis of MI with the Following.

• Plasma Creatine Kinase (CK) and Troponin:
  • Plasma levels of Creatine levels are commnoly assessed.
    • Following onset of chest pain, CK2 shows 4-8 hours and its peak activity comes at 24 and returns to baseline at 48-72 hours.
  • In infarction of the myocardium, this hybrid isoenzyme makes an apperance.
  • Troponin:
    • Troponin I is seen in hours, and has a peal in 8-28.
    • Both these are regulatory proteins involved in related ailments