Cleland Diagram & Binding Order from Lineweaver Burke Plots

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SUMMARY

The discussion centers on the Cleland diagram and binding order in Lineweaver-Burk plots, specifically focusing on the sequential and ping-pong mechanisms of enzyme reactions involving ATP and Cysteine. The participant identifies that when Cysteine is saturated, it complicates the understanding of the binding order, particularly regarding ATP's role as the first substrate despite having the highest K_m, indicating lower binding affinity. The conversation highlights the complexities of handling three substrates simultaneously and references trisubstrate mechanisms for further clarification.

PREREQUISITES
  • Understanding of enzyme kinetics and mechanisms, specifically sequential and ping-pong reactions.
  • Familiarity with Lineweaver-Burk plots and their interpretation.
  • Knowledge of substrate binding affinities and Michaelis-Menten kinetics.
  • Basic grasp of trisubstrate mechanisms in enzymatic reactions.
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  • Research the Cleland diagram for a deeper understanding of enzyme mechanisms.
  • Study the implications of K_m values in enzyme-substrate interactions.
  • Explore advanced topics in enzyme kinetics, particularly involving multiple substrates.
  • Examine literature on trisubstrate mechanisms to clarify complexities in binding orders.
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Biochemists, enzymologists, and students studying enzyme kinetics who seek to understand the intricacies of substrate binding and reaction mechanisms involving multiple substrates.

The Head
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Homework Statement
(b) Draw a Cleland diagram for the mechanism of the reaction shown above that is consistent with the following data. Briefly justify your reasoning.

(d) In light of the data in parts (b) and (c), can you conclude that ATP binds first to the enzyme? Briefly explain your reasoning.

(e) Kinetic data give the following KM values for the three substrates:
ATP KM = 1.8 mM Cys KM = 0.1 mM GlcN KM = 0.2 mM
At steady-state inside the cell, the enzyme is entirely saturated with ATP. How would you rationalize this fact in light of the above KM data?
Relevant Equations
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I am mostly focused on parts (b) and (d), which I typed out. For (b), I can tell the first pair of graphs is a sequential reaction (when Cysteine is saturated), but it appears ping-pong for the others because the slopes are roughly parallel. I know sequential has to reactants binding (so I guess that would be ATP and GlcN), and I suppose that a reaction occurs there and then maybe Cysteine binds to react with an intermediate?

But then again, if Cysteine is saturated, I don't know if that makes sense, because Cysteine should be able to bind.

Also, how do we know ATP binds first (part d)? Part (e) also confuses me, because ATP has the highest K_m, so the lowest binding affinity, but ATP is saturating the enzyme. It seems like there would be less binding for that.
 

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