Lecture 5 - Enzyme Kinetics

BIOLOGY 173, Exam 1, Lecture 5

Basic Enzymatic Reaction

Substrate and Enzyme => Enzyme-Substrate (Transition State) => Enzyme and Product

Reaction Rate

• [Amount of product formed (or substrate used)] / [Time]

• Reaction rate (velocity)

  • Product per unit of time

Factors Affecting Rate of Reaciton

• Substrate Concentration

• Enzyme Concentration

• Temperature

• pH

• Concentration of other ions

Requirements for Reaction to Proceed

• One or more chemical bonds have to break

• Other bonds have to form

• Substances must collide in specific orientation

  • Bringing electrons involved near each other

Concentration Affects Reaction Rates

• Rate of reaction INCREASES as substrate concentration increases to maximum rate (V_max)

• High concentration of reactants

  • More collisions

  • Reactions proceed faster

• Kinematics varies w/ substrate concentration

V_max

• Processing substrate to product ASAP

  • Max speed enzyme can catalyze reaction

  • When completely saturated with substrate

• Substances are not a limiting factor

K_M

• Affinity (attraction) of enzyme for its substrate

Enzyme Concentration Changes

• Less enzyme = reduced V_max

• Fewer products (in given time)

• K_M is UNCHANGED

Increasing V_max

• Increasing Enzyme Concentration

• V_max PROPORTIONAL to enzyme concentration

Enzyme Activity Regulation in Cells

• Environmental Factors

  • Temperature

  • Salts (ions)

  • pH changes

• Reversible Inhibition

  • Competitive Inhibitors

  • Noncompetitive inhibitors and accelerators

Environmental Factors Affecting Enzymes

• TEMPERATURE: increase can break hydrophobic interactions

• SALTS (IONS): can break ionic and hydrogen bonds

• pH CHANGES: can break ionic and hydrogen bonds

Reversible Inhibitors

• Competitive inhibitors

• Noncompetitive inhibitors and accelerators

Competitive Inhibitors

• DIRECTLY block enzyme’s active site

• Competes with substrate for enzyme active site

• Often resembles substrate

• Enzyme Kinematic Characteristics

  • V_max = same

  • K_M = greater (inhibitor attached instead)

    • Affinity (of substrate) = less

• At very high substrate competition, substrate OUTCOMPETES inhibitor

  • V_max remains the same

Noncompetitive Inhibitors and Accelerators

• Positive and/or negative

• ALLOSTERIC REGULATION: regulatory molecule binds away from active site

• Allosteric Effectors:

  • Can be activators or inhibitors (change enzymes conformation)

    • Activation: Allows active site to become available to substrate

    • Deactivation: Active site becomes unavailable to substrate

  • Bind at site other than active site (REGULATORY SITE)

• Alters properties of enzyme function

  • Always affects V_max

  • K_M can be unchanged, increased, or decreased

• Effect of inhibitor decreases with increasing substrate concentration (always changes K_M)

• Cannot be overcome by excess substrate (regulator binds away from active site)

Allosteric Regulation Definition

• Regulatory molecule binds away from active site

Low K_M

• Enzyme binds to substrate tightly (high affinity)

• Very efficient at converting substrate to product

High K_M

• Enzyme binds to substrate more loosely (lower affinity)

• Less efficient at converting substrate to product