Enzymes: Biochemistry Lectures

• Enzymes are taught across 12 lectures with topics covering enzymes, vitamins, hormones, minerals, amino acid metabolism, and the urea cycle
• Lippincott's Illustrated Reviews: Biochemistry (6th Edition) is the primary text
• Required reading also includes "Experimental Biochemistry for Medical Sciences Students” by T. Yigitbasi, S.S.Erdem, and P.Bozaykut and “Lehningers Principals of Biochemistry."

Learning Objectives

• The major enzyme classes for study
• The properties of enzymes for study
• Enzyme kinetics are a study objective
• The inhibition of enzymes will be studied
• Mechanisms for regulating enzyme activity will be taught
• How clinical diagnosis uses enzymes will be reviewed

Overview of Enzymes

• Enzymes act as biological catalysts, accelerating biochemical reactions
• Most enzymes are globular proteins with tertiary and quaternary structures
• Enzymes guide metabolic events by selectively channeling substrates into pathways

Enzyme Nomenclature

• Recommended names are short, for everyday use, and given a suffix of -ase which is attached to the substrate being reacted
• Systematic names classify enzymes through the International Enzyme Commission and divide enzymes into six major classes, each with subgroups, using the suffix -ase to describe the reaction

Major Classes of Enzymes

• Oxidoreductases catalyze oxidation-reduction reactions
• Transferases catalyze the transfer of chemical groups
• Hydrolases catalyze the cleavage of bonds by adding water
• Lyases catalyze the breaking of C-C, C-S, and some C-N bonds
• Isomerases catalyze the racemization of optical or geometric isomers
• Ligases catalyze bond formations with ATP hydrolysis

Enzyme Synthesis

• Enzymes are synthesized on ribosomes
• Ribosomes are attached to the rough endoplasmic reticulum
• The DNA contains the information required for enzyme synthesis
• Amino acids assemble into specific enzymes defined by DNA's genetic code

Intracellular and Extracellular Enzymes

• Synthesized and retained for use within the cell itself, intracellular enzymes are found in the cytoplasm, nucleus, mitochondria, and chloroplast
• Oxydoreductase catalyzes biological oxidation, while others in the mitochondria facilitate reduction
• Synthesized in the cell but work externally, extracellular enzymes are secreted
• An example of an extracellular enzyme is a digestive enzyme produced by the pancreas; it transports to the duodenum but, the pancreas doesn't use them

Properties of Enzymes

• They are protein catalysts that increase the rate of chemical reactions
• Located in different cellular compartments
• Enzymes exhibit varying degrees of specificity
• Absolute specificity: They act on only one substrate
• Stereospecificity: They discern between optical isomers
• Reaction specificity: They catalyze specific reaction types

The Active Site

• It is a small pocket or cleft, occupying less than 5% of the enzyme's surface, that catalyzes its functions
• Active sites, formed by protein folding, contain amino acid side chains to bind substrates
• Conformational changes occur upon substrate binding, facilitating catalysis

Enzyme Efficiency and Turnover

• Enzyme-catalyzed reactions are 10³–108 times faster than uncatalyzed reactions
• The turnover number (kcat) measures the number of substrate molecules converted to product per enzyme molecule per second
• kcat is typically 10²–10⁴ s⁻¹

Holoenzymes, Apoenzymes, Cofactors, and Coenzymes

• Some enzymes require non-protein molecules for enzymatic activity
• Holoenzymes are active enzymes that contain nonprotein components
• Apoenzymes are inactive enzymes lacking their nonprotein components
• Coenzymes and cofactors are the nonprotein components
• Prosthetic group: a coenzyme that is permanently bound to an enzyme (FAD.)
• Apoenzyme (inactive) + Coenzyme (inactive) = Holoenzyme (active)

Non-Protein Components = Cofactors

• They carry out chemical reactions that cannot be performed by standard amino acids
• Cofactors are classified as either organic coenzymes or inorganic cofactors

Inorganic Cofactors

• These molecules enhance enzyme activity
• Carbonic anhydrase requires zinc (Zn²⁺)
• Hexokinase requires magnesium (Mg²⁺)

Coenzymes: Organic Cofactors

• They are organic molecules derived from vitamins that support enzymatic activity
• Glycogen phosphorylase uses pyridoxal phosphate
• NAD+ derives from niacin, and FAD from riboflavin

Organic Cofactors & Coenzymes

• Prosthetic groups are tightly bound organic cofactors such as flavins, heme groups, and biotin
• Coenzymes, like NAD+, are loosely bound, organic cofactors

Holoenzymes versus Apoenzymes

• The enzyme without its cofactor is termed an apoenzyme
• A holoenzyme refers the the entire, functional complex, including all necessary cofactors

How Enzymes Work

• Enzymes' catalytic efficiency is explained through thermodynamics and active site processing

Thermodynamic Changes

• Only a few substances cross an activation barrier
• Slower uncatalyzed reactions occur
• Enzymes lower the energy, providing an alternate pathway for substrate conversion
• The reaction rate increases in the presence of enzymes
• The total energy of the system remains constant

Lock-and-Key Model

• Proposed by Emil Fischer in 1894
• It postulates that enzymes have rigid active sites as a hypothesis
• There is no alteration in the active site before or after a chemical reaction

Induced Fit Model

• Proposed by Daniel Koshland in 1958
• The enzyme’s active site changes shape upon substrate binding, allowing the enzyme and substrate to connect
• Enzyme kinetics studies reveal how individual steps transform substrate into product

Chemistry of the Active Site

• Transition state stabilization increases the reactive intermediate concentration
• Catalytic groups enhance the probability that the transition state is formed
• Baby = substrate
• Parent = enzyme
• Undressed baby = product

Introduction to Enzyme Kinetics

• The catalytic efficiency of enzymes is explained by two perspectives: Thermodynamic changes and Processes at the active site

Enzyme Kinetics Defined

• It studies the rate of enzyme-catalyzed reactions
• Through kinetic analysis, individual steps transforming substrate into products are revealed
• Studying enzyme kinetics can reveal the enzyme’s catalytic mechanism, its role in metabolism and how its activity is controlled and may be inhibited

Factors Affecting Reaction Velocity

• Substrate concentration is a factor to consider
• A reaction rate is the amount of substrate converted to product per unit of time (µmol/min)
• Reaction velocity increases with substrate concentration until reaching maximum velocity
• Most enzymes follow Michaelis-Menten kinetics, displaying a hyperbolic shape, whereas allosteric enzymes show sigmoidal curves

Other Factors Affecting Reaction Velocity

• Temperature: Enzymes for humans show stability up to 35–45°C.
• pH: Enzymes exhibit optimal activity at pH 5–9.

Michaelis-Menten Kinetics:

• Enzymes reversibly combine with a substrate to form an ES-complex that subsequently yields a product and regenerates the free enzyme
• E + S ↔ ES → E + P (k₁, k₋₁, k₂)

Michaelis-Menten Equation

• It describes how the reaction velocity changes with substrate concentration
• Vo = (Vmax [S]) / (Km + [S])
  • Vo is initial reaction velocity
  • Vmax is the maximum velocity
  • Km is the Michaelis constant = (k₋₁+k₂)/k₁
  • [S] is the substrate concentration

Assumptions of Michaelis-Menten Kinetics

• Relative concentrations of E and S, where [S] >> [E]
• Steady-state assumption: [ES] doesn’t change over time
• Initial velocity is measured when enzyme and substrate are initially mixed, and the reverse reaction is ignored; the rate of ES formation equals breakdown

Km Implications

• Km reflects enzyme affinity to substrate
• Km = [S] at ½ Vmax
• Inverse relationship between Km and affinity exists
• Reduced enzyme concentration decreases V₀ and Vmax
• At [S]<<Km, rate = [S] means the reaction is first order
• At [S]>>Km, rate is independent of [S] means the reaction is zero order

Lineweaver-Burk Plot

• It calculates Km and Vmax, used when gradual slopes are not able to do so
• When V₀ is plotted against [S], it is not always possible to determine when Vmax has been achieved due to gradual upward slope.
• Calculated using the formula 1/Vo = 1/Vmax + Km/Vmax (1/[S])

Enzyme Inhibition

• It is defined as preventing enzyme processes by inhibitors, and any substance that reduces enzyme reaction velocity acts as an inhibitor

Types of Inhibition

• Irreversible inhibitors bind through covalent bonds
• Reversible inhibitors bind through non-covalent bonds
• Dilution causes dissociation of reversibly bound inhibitors

Irreversible Inhibition

• Inhibitors covalently attach to the enzyme and make it lose catalytic activity completely
• The enzyme must synthesize molecules to restore function

Irreversible Inhibition Examples

• Aspirin, inhibits inflammation by covalently modifying key enzymes

Competitive Inhibition

• Inhibitors and substrates compete for the active site
• Formation of the ES complex is reduced while forming a new EI complex
• Increased [S] reduces the inhibitor effect as Vmax can be attained through enough substrate
• Inhibitors increase Km, and a Lineweaver-Burk plot shows an intersection between inhibited and uninhibited reactions on the y-axis at ½ Vmax

Statin Drugs

• They inhibit HMG-CoA reductase within cholesterol synthesis (e.g. Lipitor, pravachol)
• Analogs of natural substrates also compete effectively for that enzyme

Noncompetitive Inhibition

• A type of inhibition where the inhibitor binds to allosteric site instead of the active site and that the enzyme is unable to be overcome by increasing [S]
• Inhibitors don't affect substrate binding, so Km is unchanged
• Vmax decreases because of the Inhibitors

Common Enzyme Inhibitors

• Half of the commonly prescribed drugs in the US act as enzyme