A question about Km's of Firefly Luciferase

Eshi

So heres the background, I am currently doing research on one of the luciferase proteins. The luciferase protein creates bioluminescence using the substrates ATP and Luciferan. Its a two step process... but the general sense is that u can use the light to measure the activity of the protein as a unit of the amount of protein in the solution. What this means is that u can measure the km by using the relative values obtained by the light emitted by the protein. This process measures the 2 step reaction as a whole, and allows for an accurate km for the two substrates Luciferan and ATP.

My issue is that we created a mutant in which the km for luciferan was much lower than wild type. However, when we tried to do the ATP km, and the km values continually do not make sense, the bioluminescence is greatly reduced, even when using the same concentrations.

My boss ssaid possibly its the order of reagents, but why would that reduce the bioluminescence?

P.S. I am only a freshman in college, so my knowledge of biochem is limited. WHere should i go to learn more about this type of thing? or could someone help?

Ygggdrasil

Are you sure that the mutation affects only the K_m of the reaction and not the catalytic rate (k_cat, reflected by a change in V_max of the enzyme)?

epenguin

What does not make sense is your sentences so it is difficult to make suggestions. What actually are the observations? preferable with summary and diags.

By 'order of reagents' does your supervisor mean order of adding the reagents in an measurement experiment or order of addition of substrates to enzyme in the reaction mechanism?

That said, Ygggdrasil's is the most obvious first guess.

Moonbear

Unfortunately, I share ePenguin's confusion in trying to understand the sentence describing your experiment and results.

However, since you are a freshman, I assume you are doing this as an internship type project to learn more about research. If so, you should be able to ask your "boss" if you don't understand his/her explanation. S/he knows what you've been trying to do better than we do, so should be able to explain her/his reasoning to you, or offer you some resources to learn some of the basics you need to understand the subject.

Otherwise, if you want help here, you're going to need to provide a lot more information, such as what reagents you're using, the order you're using them, the conditions of the reaction, etc. Importantly, what are your controls? You said the reaction is a two-step process, and it sounds like you mean that luciferan and ATP are each substrates for the reaction in wild type, but not that they necessarily need to both be present at the same time. Is that correct? Do you first react with luciferan, then ATP, or vice versa? Or do both need to be present together? How do you know your mutant is capable of using both substrates? If it uses luciferan, but you have lower luminescence in the presence of ATP when comparing with wild type, how have you ruled out that the mutation isn't leading to a defect in ability to use ATP as a substrate?

VirtualAgeJ

Dear Eshi,
Your boss might be on to something as ATP plugs into the active on top of luciferin (LH2). If you enhance the affinity (remember the real measure of that is Ks - possible with quenched Trp fluorescence experiments) for LH2, you might upset the reaction for ATP if LH2 (added first in LH2 Km measurements) alters the site somehow. Even just the binding equilibria and kinetics are complicated. I'm not too hot on this, ask your boss. When you measure ATP Kms, you add ATP first and saturating LH2 after which has to fit in the site. However, this may not be the case if merely the Km for ATP is higher in your mutant than before and you are not getting to Vmax - make sure to saturate starting from ca. 5mM ATP.

Read up on Luc kinetic models (Ugarova, Gandelman etc from Moscow). There are points on this very issue (i.e. addition order of substrates) in there 1990s papers.