Enzymes

Enzymes

• Enzymes are biological catalysts responsible for chemical reactions that maintain life in metabolism

  • Allow cells to exert kinetic control over thermodynamic potential

  • Almost all proteins (excludes class of RNA catalysts)

• Biocatalysts vs chemical catalysts

  • Both increase the rate of reaction

  • Have no effect on ΔG or Keq

  • Speed up the rate at which a reaction approaches equilibrium

• Properties

  • High reaction rates

  • Catalyze reaction a physiological conditions (dilute, aqueous, pH, temperature, pressure)

  • Have a high degree of specificity and very few side reactions

  • Can be regulated (amount, activity, binding of ligands, covalent modification)

  • Reactions can be stereospecific

• The transition state

  • ΔG- change in Gibbs free energy for a complete reaction

    • Overall free energy change for a reaction is related to the equilibrium constant

  • ΔG‡- change in Gibbs free energy at the transition state of a reaction

    • Free energy of activation for a reaction is related to the rate constant

• Enzymes can enhance rate by 10⁵ to 10¹⁷

How do enzymes work?

• Before a substrate can become a product it must have a minimum energy to pass through a transition state

• Enzymes act by lowering the activation energy

  • Only the rate of the reaction differs

Enzyme Classes

Cofactors and Coenzymes

• Nonprotein components of any enzyme that are required for enzyme function

• Cofactors- includes organic and inorganic ions like metals, serve as transient carriers of specific atoms or functional groups

• Coenzymes- organic molecules (usually activated vitamins)

• Metal cofactors

• Organic cofactors

• To carry electrons in metabolism the vitamin must be activated by adding an amide, ribose, phosphates, and adenosine

• Tightly bound cofactors

  • AKA prosthetic groups

  • Strongly attached with their protein via covalent bonds

  • Holoenzyme: enzyme with its prosthetic group attached

  • Apoenzyme: enzyme with its prosthetic group removed

Specificity

• Enzymes selectively recognize proper substrates over other molecules

• Lock and key binding model: same set of noncovalent interaction that enable a protein to fold are involved in stabilizing the interaction between substrate and enzyme

• Induced fit: more common, enzyme active site adapt its structure to interact with substrate and transition state

How to measure enzyme activity

• Purify enzyme

• Identify an unmeasurable reaction

• Mix enzyme and large excess of substrate in a buffered system

• Monitor appearance of product or disappearance of substrate

• Calculate velocity/rate of reaction

• Vmax- maximum velocity of the reaction under the conditions studied

• Assume enzyme substrate complex is in rapid equilibrium with free enzyme

• Break down of ES to form products is assumed to be slower than

  • Formation of ES

  • Breakdown of ES to reform E and S

• Km is a constant derived from rate constants and an estimate of the dissociation constant of E from S

  • Small Km means tight binding and high Km means weak binding

• Kcat- turnover number, number of substrate molecules converted to product per enzyme molecule per unit of time when E is saturate with substrate

• Catalytic efficiency- kcat/Km

  • Apparent second order rate constant

  • Measure how the enzyme performs when S is low

  • Upper limit is the diffusion limit, the rate at which E and S diffuse together