EFB325 Cell Physiology

Enzymes most commonly are proteins (name them by adding -ase, i.e. cellulase), but RNA can also catalyze reactions=ribozymes

The reactant in an enzymatic reaction=substrate

• substrate binds to an active site-usually very specific pocket within the folded shape of a protein
• protein changes shape a little when substrate binds, stresses bonds, puts atoms adjacent to reactive amino acids or prosthetic group (like an Fe atom) in the protein

Increased temperature increases the rate of the reaction (due to increased energy), but ...

• enzymes have an optimum temperature (before they are denatured)
• and an optimum pH (at which R-groups are protonated or deprotonated)

Enzymes are catalysts:

• they participate in the reaction, but are not changed when it is completed
• do not affect the balance of the equilibrium, only the rate at which equilibrium is reached
• specific for the reaction (unlike adding heat); rate=10⁸ - 10¹⁰-fold faster
• reduce the activation energy by bringing the reactants into position that facilitates reaction (form a transient complex)

Enzyme kinetics

(discovered by Michaelis and Menten)

Enzymes catalyze reactions by binding to the substrate and facilitating the reaction, thus lowering the activation energy

• the enzyme (E) and substrate (S) form a transient intermediate complex (ES)
• the enzyme can stabilize the transition state of the reaction, which is the highest energy point in the system, this lowers the activation energy

There are three rates to consider in the reaction

• k₁=rate of substrate binding to enzyme to form the ES complex (rate of E+S->ES)
• k₂=rate of substrate dissociation from the ES complex (rate of ES->E+S)
• k₃=rate of the reaction and product dissociation (rate of ES->E+P)=the turnover number of that enzyme

Michaelis-Menten equation is:

V = V_max ([S] / [S] + K_M)

• V = the rate of product formation
• [S] = substrate concentration
• K_M = the Michaelis constant = ( k₂ + k₃ ) / k₁

When analyzing enzyme kinetics, we usually hold the amount of enzyme constant

• at low substrate concentrations, the rate of the reaction will increase linearly with an increase in substrate
• BUT, with increasing substrate concentration as the binding sites on the enzymes become saturated with substrate, the rate of the reaction will approach a maximum (V_max) {at high [S], V approaches k₃[E]}

The substrate concentration at which the formation of ES is equal to the breakdown of ES (a steady state) is defined as the Michaelis constant (K_M)

• at the K_M, V=1/2V_max
• usually the K_M represents a measure of the binding affinity of the enzyme, a high K_M means that the enzyme has a low binding affinity and low K_M means that the enzyme binds to the substrate very tightly

Regulation

Reactions in the cell are regulated by regulation of enzyme activity

• the amount or compartmentation of enzyme can be regulated
• more commonly, the catalytic activity of existing enzyme molecules can be varied

Enzyme activity is sensitive to temperature and pH, which can alter the chemical interactions responsible for maintaining 3-D structure and substrate binding

Enzymes often bind another molecule in addition to the substrate

• binding at a regulatory site
• may act as an inhibitor or as an activator
• binding of another molecule often causes a slight change in the shape of the enzyme, altering its activity=allosteric regulation
• often the final product of a pathway will act to inhibit an enzyme that catalyzes an early step in the pathway=feedback inhibition

Addition or removal of a phosphate group to one or more amino acids (usually Ser, Thr, Tyr, or His) in an enzyme can alter its activity

• addition of a phosphate group=phosphorylation and is catalyzed by enzymes called kinases
• removal of a phosphate group=dephosphorylation and is catalyzed by enzymes called phosphatases

Thus, the activity of an enzyme (even a kinase or phosphatase) can be regulated by the activity of another enzyme (a kinase or phosphatase)

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