Study Notes on Enzyme Kinetics with Multiple Substrates

Enzyme Kinetics with Multiple Substrates

Introduction to Bisubstrate Enzymes

• Focus on enzymes that bind two substrates, as discussing enzymes that bind three or more becomes increasingly complex.

• Distinction in kinetic mechanisms based on the binding order of substrates and release order of products.

Kinetic Mechanisms of Bisubstrate Enzymes

• Two primary mechanisms for enzymes with two substrates:

  • Sequential Mechanism (Bi Bi Mechanism)

  • Forms a ternary complex.

  • Can be either random or ordered.

  • Ping Pong Mechanism

  • No ternary complex is formed.

Sequential Mechanism

• Ternary Complex: Complex formed when both substrate molecules are bound to the enzyme simultaneously.

• Random Sequential Mechanism:

  • Substrates A and B bind without any specific order.

  • Products can also be released in any order.

  • Example: Creatine kinase.

  • Cleeland Nomenclature:

    • Free enzyme: E

    • Substrates: A, B

    • Products: P, Q

  • Potential pathways for binding:

    • A binds first, followed by B, forming the enzyme-substrate complex.

    • B binds first, followed by A, still forming the enzyme-substrate complex.

  • Product release pathways:

    • P released first, followed by Q; or Q released first, followed by P.

• Lineweaver-Burk Plot Analysis:

  • Holding concentration of A constant and varying B.

  • Intersection of lines occurs left of the y-axis, indicating a ternary complex formation.

Ordered Sequential Mechanism

• In contrast, Ordered Mechanism:

  • A must bind before B.

  • Steps:

  • Free enzyme binds substrate A to form an enzyme-substrate complex (E-S complex with only A).

  • Binding of A creates conditions for B to bind, forming the ternary complex.

  • Conversion of substrates to products and sequential release (P before Q).

• Example: Alcohol dehydrogenase.

• Lineweaver-Burk Plot:

  • Ternary complex formation evident but intersection does not touch the x-axis; it is to the left of the y-axis.

  • Holding concentration of A constant while varying B shows altered intersection characteristics compared to random mechanisms.

Comparison of Sequential Mechanisms

• Distinguishing Ordered from Random Mechanisms:

  • Not straightforward at the start of analysis; results from analysis will indicate the type.

  • Random mechanisms manifest intersections at the x-axis; both types generally show intersection left of the x-axis.

Ping Pong Mechanism

• Different from sequential mechanisms as it represents a Double Displacement reaction.

• Steps:

  • Formation of an enzyme-substrate complex with the first substrate.

  • Results in an altered enzyme (E becomes F); substrate A is converted to product P and is released.

  • An altered enzyme is now available for interaction with a second substrate, which binds, forming an altered enzyme-substrate complex.

  • This leads to regeneration of the initial enzyme and production of the second product.

• Example: Aminotransferases.

• Characteristics of Lineweaver-Burk Plot:

  • No ternary complex forms, so the lines do not intersect and instead remain parallel.

  • Despite varying substrate concentrations, the slopes remain consistent across different plots while the intersects differ, resulting in parallel lines in the graphical representation.