Biochemistry: Enzymes and Kinetics

Enzymes

• Enzymes are proteins that catalyze biochemical reactions.
• Enzymes lower the activation energy for both forward and reverse reactions.
• Enzymes do not change the overall free energy (ΔG) change of a reaction.

Rate vs Direction of Biochemical Reactions

• Free energy is the energy available to do work as a reaction approaches equilibrium.
• ΔGrxn = ΔΗrxn - TAS
• ΔGrxn < 0 for spontaneous reactions.
• ATP is thermodynamically unstable but kinetically stable. This is shown in a free energy diagram for ATP hydrolysis. ΔG° = -30.5 kJ/mol.

Biochemical Kinetics

• Rate = k [A][B]², where k is the rate constant.
• Rate constant, k, is defined by the Arrhenius equation and includes the temperature and activation energy (Ea = ΔG++).
• Ea = Ae^-Ea/RT, where A is the frequency factor, R is the gas constant, and T is the Kelvin temperature.

Catalysts

• Catalysts increase the rate of reactions toward equilibrium by lowering ΔG++.
• Enzymes can provide enhancements of 10⁶ to 10¹⁴ in reaction rates.

Enzyme Classification

• Oxidoreductases: catalyze oxidation-reduction reactions.
• Transferases: transfer functional groups.
• Hydrolases: catalyze hydrolysis reactions.
• Lyases: catalyze group elimination to form double bonds.
• Isomerases: catalyze isomerization reactions.
• Ligases: catalyze bond formation coupled with ATP hydrolysis.

Enzyme Nomenclature

• Enzymes usually end in "-ase."
• Common names are often ambiguous; systematic names are established by the enzyme commission of IUBMB (e.g., carboxypeptidase A, systematic name: peptidyl-L-amino acid hydrolase EC.3.4.17.1).

How do enzymes lower ΔG^‡?

• Binding and orientation of substrate(s) to increase reactivity and proximity of chemical groups.
• Preferential binding of X⁺.
• Provide an alternative pathway with a lower ΔG^‡.

Chemical Bases for Enzymatic Catalysis

• Acid-base catalysis (H⁺ transfer)
• Covalent catalysis (transient covalent bond)
• Metal ion catalysis
  • Transition metal ions in active site play a role in catalysis (Fe, Cu, Zn, Mn, Co).
  • Contributions to electrostatic catalysis.
  • Increase reactivity of H₂O by polarization.
  • Redox reactions.
  • Electrostatic effects (stabilization of X⁺, binding, and orientation of substrate S)
  • Electron transfer (redox)
  • Polarization of H₂O (e.g., carbonic anhydrase)
  • Charge-shielding on substrate (e.g., Mg-ATP).

Specific Example: RNase

• Two key histidine residues in the active site: His 12 (base) and His 119 (acid).
• An example of a hydrolysis reaction.

Enzyme Kinetics

• Michaelis-Menten Equation describes the reaction rate of non-allosteric enzymes
• Rate equation: v = V_max[S]/ (K_M + [S])
• V_max = maximum velocity
• K_M= (k_-1 + k₂)/ k₁ or rate of dissociation of ES

Significance of K_M

• K_M is the substrate concentration at which the reaction rate is half-maximal (V_max/2)
• related to the equilibrium binding affinity of E for S

Catalytic Constant, k_cat

• k_cat is a first-order rate constant that represents the maximum rate of a substrate turnover in an enzyme catalyzed reaction.
  • k_cat = V_max/E_T , (where E_T is the total enzyme concentration)

Specificity Constant, k_cat/K_M

• A measure of catalytic efficiency. A higher rate generally means better efficiency

Two-Substrate Reactions

• Sequential mechanism
• Both substrates bind to the enzyme, producing a ternary complex, before any product is formed.
• Ping-pong mechanism
• One substrate binds, then the first product is released before the second substrate binds.

Allosteric Enzymes

• Multi-subunit proteins (multiple catalytic and regulatory subunits).
• Subunit interactions stabilize low-affinity (T) state.
• Allosteric effectors (modulators) can be positive or negative.
• Substrate binding can show positive cooperativity.

Example: Aspartate Transcarbamoylase

• First step in pyrimidine biosynthesis.
• Complex of six catalytic and six regulatory subunits (C₆R₆).
• Allosteric effectors: substrates (+), CTP (-), ATP (+).

Covalent Modifications

• Phosphorylation, adenylation, acetylation, myristoylation are common covalent modifications that produce allosteric-like changes.

Regulation of Enzyme Activities

• Rapid: noncovalent modification (competitive inhibition, substrate/effector binding).
• Slower: covalent modifications (phosphorylation), degradation and synthesis of mRNA/polypeptides.

Enzyme Inhibition

• Irreversible inhibitors: inactivators (typically very reactive electrophiles)
• Reversible inhibitors: bind noncovalently and reversibly.

Different types of Enzyme Inhibition

• Competitive Inhibition
• Uncompetitive Inhibition
• Mixed Inhibition
• Noncompetitive Inhibition

Explore the fascinating world of enzymes and their role in biochemical reactions. This quiz covers key concepts such as the relationship between free energy, reaction rates, and catalysts. Test your knowledge on how these proteins function and impact biochemical processes.