Bi-substrate Enzyme Kinetics Sequential 1. ordered 2. random Ping-pong

Equations for Bi-substrate Kinetics 1/v Vmax[A][B] v = Ka[B] + Kb[A] + [A][B] 1/[A] [B] Vmax[A][B] 1/v v = [A][B] + Ka[B] + Kb[A] + KaKb 1/[A]

Adenylate Kinase Kinetic Pathway Adenylate kinase displays a random ordered kinetic mechanism. In this case, the two substrates are bound randomly, and are in equilibrium with the “ternary complex” (E•MgATP•AMP). As in our derivation, this necessitates that the off rate for each of the substrates is less than the forward rate constant for the chemical step. This allows us to replace Km with Ks. However, it would not be incorrect to use Km values. Below is typical shorthand notation for kinetic schemes.

Nucleoside Diphosphate Kinase Nucleoside diphosphate kinase (NDP Kinase) catalyzes the transfer of the terminal phosphoryl group of ATP to a nucleoside diphosphate. NDP Kinase displays a steady state kinetic pattern that is distinctly different from that of adenylate kinase. If one substrate is varied while the other is fixed at several different concentrations, a family of parallel lines is obtained by Lineweaver-Burk analysis. This is reminiscent of a Ping-Pong reaction.

UDP-Glucose Pyrophosphorylase Steady State kinetic equation is similar to adenylate kinase. Therefore Lineweaver-Burk plots cannot distinguish the two forms of sequential reactions. Must do product inhibition studies. Stereochemistry indicates inversion; however, incubation of the enzyme with radiolabeled UTP, followed by gel-filtration shows a radiolabeled intermediate. Be careful! This is because UTP or UDP-glucose binds very tightly to the enzyme. In fact, the enzyme is isolated with UTP and UDP-glucose tightly bound, and will catalyze an exchange reaction, which is characteristic of Ping-pong reactions.

Ping-Pong Reaction

Galactose-1-P Uridylytransferase