Hi Emma !!!

I have found something and it might help you. Please refer to the following paper:

Article Enzymes with Two Optimum pH Values.

Thanks.

Hello, the question is a rather new phenomenon described in some publications.

See attached info that may be useful for you.

I think the multiple optimal pH values can be a result of both, which can cause the conformational changes in both enzyme and substrate depends on the pH value of the reaction mixture.

Goog luck!

My guess is that it is because somehow change of pH affecting the conformation of substrate also. So, pH is affecting both the enzyme and substrate. The combined result is enzyme's activity seems to be highest at two different pH

You are using two different buffer substances, making the results difficult to compare. In addition, there is a gap between the data. You should either use a buffering substance that offers a wide pH-range (like citrate), or buffer mixtures with wide pH-range (PACT-buffers doi:10.1107/S0907444903029640, Britton-Robinson doi:10.1039/JR9310001456, McIlvaine https://www.jbc.org/content/49/1/183.short).

In in vitro studies, this behavior can occur of "appearing" two activity peaks as a function of pH. As Dr. Buxbaum indicates, it is important to use the same buffer system for all pHs, one option is Stauffer's Universal buffer. However, the recommendation for studies of the physiology of the species and the quantification of enzymes is that it should work under physiological conditions. In this particular case at physiological pH.

Stauffer's buffer (1989) actually appears to be Teorell & Stenhagen: "Ein Universalpuffer für den pH-Bereich 2.0 bis 12.0", Biochem. Z. 299 (1938) 416-9 (boric, citric and phosphoric acid). Unfortunately, Biochem. Z. (later Eur. J. Biochem.) is not available electronically, but a well-stocked library should have it.

this case is encountered when the enzyme catalyzes a reversible reaction, an enzyme having several substrates or an enzyme immobilized in different support

Hi all, thank you for your responses. I've read the "Enzymes with two pHs" paper, which unfortunately refers to different substrates, instead of two peaks on one substrate, which is what I'm experiencing. Furthermore, this is a new enyzyme and we don't yet know the physiological pH at which it acts in vivo.

Thank you to Hector Nolasco and Engelbert Buxbaum for the suggestion of Stauffer's universal buffer! I will have to let my supervisor know.

Ouahiba Harrieche what do you mean "immobilized in different support"?

an immobilized enzyme is an enzyme attached to an inert and insoluble support by covalent or non-covalent bonds. an enzyme may be attached to different supports depending on the desired application. this binding can modify the optimal pH of the enzyme

Ouahiba Harrieche I don't this this is an immobilized enzyme.

Emma Quinlan Hi, the double pH may be because the substrate and enzyme respond differently to pH changes. While the substrate may become charged at one point (and this may affect it's binding and/or the reaction), the enzyme may be affected at another pH point and that in turn may again affect binding and/or the reaction.

Alternatively, both changes may be due to the enzyme if two changes occured.

It's not very big peak, but if you look at Fig 5C

Article Biochemical Characterization of Cytokinin Oxidases/Dehydroge...

you'll see that AtCKX1 with iPR and Q0 as cofactor has sort of two pH optima, one at 4.5 and the other around 6.5.