I share your frustration with students who do not grasp the reversibility of enzyme reactions, but it is actually a misunderstanding of the Michaelis-Menton method (more appropriately Victor Henri’s equation, since he derive it 10 years before MM used it, and they him full credit for the derivation in the original MM publication). The Henri-Michaelis-Menton equation uses initial rates so that we can avoid 1) product inhibition, 2) substrate depletion and 3) the reverse reaction you are concerned with. That is why the k2 step appears to be irreversible…it is under the initial rate conditions. Henri (and Michaelis & Menton) were primarily concerned with the problem of product inhibition, which was a big problem with their invertase. I hope that helps you some with the utter frustration you feel. Best of luck with future endeavors. 

You're touching on a very sore spot for me here. The bane of my existence for quite a few years was the body of research surrounding a particular enzyme which catalyses a class of reactions with near-zero change in free energy. Despite this, almost two decades of papers in the literature have run on the belief that the reaction is irreversible - an idea that not only flies in the face of thermodynamics, but badly distorts the interpretation of its biology. Such misconceptions can prove exceedingly difficult to dispel. Of course, it's fairly easy to see where such ideas come from - irreversibility of the reaction is a key assumption in the derivation of the Michaelis-Menten equation, and I'm quite sure that many students in biochemistry are never properly introduced to the concept of reversible reactions.

Cheers,

Tristan

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From: CCP4 bulletin board <[log in to unmask]> on behalf of R. Michael Garavito <[log in to unmask]>
Sent: Tuesday, 2 February 2016 3:02 AM
To: [log in to unmask]
Subject: Re: [ccp4bb] Michaelis–Menten and lineweaver burk

 

Jacob,

I agree completely, and I also feel that even Michaelis-Menton kinetic analysis is obsolete. Doing a pseudo-M-M kinetic analysis (varying the max concentration of the 2nd substrate) will give you a lot more information than trying the make a 2 substrate enzyme behave like a single substrate enzyme.  Or dumping all of your kinetic data for 2 substrate enzyme into Origin to model what you believe is the right rate equation is also preferred, but not easy.  

However, Michaelis-Menton kinetic analyses are simple to do, which is why so many people do them, but it also too easily abused.  L-B plots, whether done by hand or done with a fitting program, are easy to understand for undergraduates, but they obscure a lot of basic enzyme behavior (try dealing with an enzyme that shifts between a sigmoidal and hyperbolic substrate saturation behavior with temperature).  The Hill analysis is an equally big problem particularly with multiple enzyme-substrate/enzyme-intermediate equilibria.   

My pet peeve is that many young researchers, like most undergraduates, just believe that an M-M kinetic analysis works in all cases and blindly trust the modeling (particular since multi-parameter fitting became so readily available).  Few investigate whether one’s assay design and methodology are valid for analyzing a particular enzyme in the first place.  That’s why I will stay with structural analysis rather than doing more enzymology.

Cheers,

Michael

On Feb 1, 2016, at 11:31 AM, Keller, Jacob <[log in to unmask]> wrote:

I thought L-B was inferior but mathematically roughly equivalent and made it easier to use a physical ruler graphically to find the intercept, and is therefore obsolete in the face of computer fitting software. And I just looked at Wikipedia, which says the same.

 

Also, the Hill equation is preferable for substrate binding analysis, since it includes a measure of cooperativity (Hill coefficient.)

 

Jacob