Protein Functions: structural, enzymatic, transport

• Proteins have various functions including structural, enzymatic, gas transport/storage, nutrient, antibodies, hormonal, and mechanical.

Structural

• Examples include: keratin, collagen, and fibroin

Enzyme

• Enzymes carry out chemical reactions, and are the most numerous type of protein.
• Highest number of proteins
• Occur in 1000s of rxns

Gas transport/storage

• Examples include: hemoglobin and myoglobin

Nutrient

• Ovalbumin is the main protein in egg white.
• Casein is the predominant protein in milk.

Antibodies

• IgG proteins recognize foreign objects and pathogens in vertebrate immune systems.

Hormones

• Peptide hormones include oxytocin and vasopressin.
• Protein hormones include insulin.

Mechanical work

• Facilitated by myosin and actin aka molecular motors

Enzymes as Catalysts

• Catalysts increase the rate of a chemical reaction without undergoing permanent chemical change.
• Catalysis is the acceleration of a chemical reaction by a catalyst.
• Enzymes are the most efficient catalysts known.
• Without catalysts, most biological reactions would occur too slowly to sustain life.
• Increase reaction rate from 1 to 1 x 10^20

Enzyme Reaction Rates

• Reactions require activation energy (ΔG°‡).
• Activation energy is the energy input needed to initiate a reaction.
• Free energy change of a run (ΔG°) is the energy change between reactants and products.
• ΔG can be calculated by knowing the energy stored in reactants and the energy given off.
• Transition state is the maximum point on the reaction curve where energy is maximal and atom arrangement is optimal for product formation.
• A reaction that has reached the transition state will always proceed to product.
• ΔG°‡ represents the amount of energy to bring reactants to the transition state.
• Enzymes lower the activation energy (ΔG°‡), thus increasing the reaction rate.
• Enzymes lower activation energy by changing the reaction mechanism to require less energy.
• Enzymes find an easier route for reaction.
• Uncatalyzed reactions have a higher activation energy than catalyzed reaction- slower reaction rate

Enzymes and Transition State

• Enzymes have no affect on the transition state
• Enzymes can affect the activation energy
• The free energy change of uncatalyzed and catalyzed reactions are the same.
• Hydrogen peroxide can be catalyzed by either platinum or the enzyme catalase.
• Catalase 23501x faster than Pt

Enzyme-Substrate Binding

• A substrate is a reactant that forms a complex with the enzyme.
• Transition-state species is the reaction intermediate present at the transition state.
• The active site is the area of the enzyme with amino acids essential for enzymatic activity.
• The active site is the substrate binding site, located in a cleft or crevice, and is where the reaction occurs.
• The interaction between enzyme and substrate involves noncovalent interactions, is highly specific.
• Substrates interact with side chain and backbone groups of active site amino acids.

Models of Enzyme-Substrate Binding

• Lock and Key
• Induced Fit

Lock-and-Key Model

• Substrate binds to active site that has complementary shape.
• It does not account for conformational change in enzyme.

Induced-Fit Model

• This model better accounts for conformational changes in the enzyme after substrate binding.

• Enzyme changes shape upon substrate binding, which allows full binding of substrate

• This model is favored over the Lock and Key Model

• E + S→ES→EX‡ → E + P

Comparing Enzyme-Substrate Binding Models

• In the lock-and-key model, if the ES complex is a perfect fit, the activation energy would be too large to overcome.
• By not having a perfect fit of ES complex, the activation energy is kept low so reaction can proceed.
• The lock-and-key model has a lower free energy state compared to ES intermediate.
• The difference between ES and transition state is too great to overcome in the the lock-and-key model, as free energy is increased.
• The induced-fit model means the free energy is not so low and the differences between ES and transition state is not so large.
• Once substrate is bound, the transition state is formed.
• Substrate is arranged in correct orientation with respect to enzyme atoms.
• Allows for bonds to be broken and new ones formed, then product to be formed
• Product released

Leonor Michaelis and Maud Menten

• Leonor Michaelis was a German biochemist.
• He worked at many universities and did groundbreaking work on enzyme kinetics at Berlin University in 1913.
• Maud Menten: Canadian biochemist
• At the time women could not get Ph.D. in Canada
• Completed medical doctorate at University of Chicago
• Moved to Berlin University in 1912 to work with Michaelis, and obtained Ph.D. in 1916
• They studied kinetics of enzyme-substrate interaction (measurement of rates of runs).

Measuring Rates of Reactions

• Mix enzyme and substrate in a buffer at a proper pH in a test tube.
• Allow reaction to take place, and take samples at time points.
• Then measure amount of product formed per time [P]/time=v.
• Repeat reaction using different substrate concentrations [S] and graph results.
• The rate of reaction increases as [S] increases.
• As [S] reaches higher levels, the reaction rate changes less until maximum rate is reached.

Michaelis-Menten Kinetics

• At 1/2 of the maximum velocity, the [S] is equal to Km.
• Km is an inverse measure of the affinity of an enzyme for its substrate.
• Lower Km means higher affinity.
• Km is the [S] at which 50% of the enzyme is occupied by substrate.
• Maximum velocity is theoretically unattainable due to asymptotic reaching at infinity of [S].

Lineweaver-Burk Plot

• Hans Lineweaver: American biochemist
• Worked as a graduate student under Burk
• At Department of Agriculture laboratory in Washington, D.C.
• Dean Burk: American biochemist
• Entered UC Davis at age 15
• In 1934 published paper with Lineweaver-Burk describing how to determine Km and maximum rate.
• It represents a most frequently cited paper in biochemistry.
• A straight line is obtained if 1/V and 1/[S] (reciprocal) are plotted.
• The slope of the curve is then used to determine Michaelis-Menten data.

Blood Sugar and Glycolysis

• Hexokinase: Enzyme is found in most tissues, and converts glucose to glucose-6-phosphate (G6P)
• Production of G6P is the first step in glycolysis (break down of glucose for energy)
• Glucokinase: Variant of hexokinase in liver
• In the G6P liver, converts to glycogen (polymer of glucose broken down into glucose) w/ low blood glucose levels.
• Blood [glucose] = 5 mM
• Low blood [Glucose], ≤5mM
• Liver: glucokinase not active, and glucose is not stored as glycogen
• Muscle: hexokinase active, and G6P produced by glycolysis
• Blood [glucose] after meal = 10mM
• High blood [glucose], ≥10mM (after meal)
• Liver: glucokinase active, G6P produced, and glucose stored as glycogen
• Muscle: hexokinase active, and G6P produced for glycolysis
• blood [glucose]↓Glucokinase inactive
• blood [glucose]↑Glucokinase active

Enzyme Inhibitors

• Enzyme inhibitors interfere with enzyme activity and slow reactions.
• Reversible: Bind to enzyme and inhibit run (inhibitor can be released and enzyme left in original condition)
• Irreversible: Binds to enzyme and makes it inactive and enzyme will not return to original condition
• Enzyme is not released

Reversible Enzyme Inhibitors Types

• Two types: competitive and noncompetitive inhibitors
• Competitive inhibitors are structurally similar to substrate.
• Binds to an active site and prevents substrate from binding to enzyme, competing for an active site with the intended substrate
• Substrate or inhibitor can bind enzyme, but not both at same time.
• Noncompetitive inhibitors bind at a site other than the active site and enzyme, but the presence of such other inhibitor binding causes a conformational change in structure and renders it inactive even if the structural substrate can still bind.
• Both substrate and inhibitor can bind at same time.

Affect of Competitive and Noncompetitive Inhibitors on Enzyme Kinetics

• Competitive: more substrate is needed to reach same velocity rate. And competitive inhibitors can be overcome with sufficiently high amounts of substrate.

• Affects on enzyme kinetics (based on Lineweaver-Burk plot)

• Changes the slope of the plot, and changes Km (x-intercept)

• Does not change maximum velocity(y-intercept)

• Noncompetitive inhibitor

• Substrate can still bind fully to enzyme, but inhibitor does not interfere

• Noncompetitive: because noncompetitive inhibitor and substrate are not competing for active site, cannot overcome inhibitor