Substrate selection for bifunctional enzyme

[Boopathi]

We are characterizing the Beta-Galactosidase from a hot spring bacteria. While assaying for B-Galactosidase using o-Nitrophenol Galactopyranoside we found that it also has Glucosidase activity. I looked for Glucosidase substrate and found that p-Nitrophenol Glucopyranoside is the commonly used substrate for B-Glucosidase. I want to check the Km and Vmax for both the substrates. But I could not find both of the substrate either with o-nitrophenol or p-nitrophenol. If I want to compare the enzyme actvity do I need to use the similar substrate or I can use different substrate, kindly clarify me....

[Jens]

I think you cannot compare the values, since the o-NO₂ group (in contrast to the p-NO₂) is sterically too close to the hydrolized bond and therefore to the active center of the enzyme. So the difference might at least partially be caused by the different NO₂ group position and not by the sugar.

 

(by the way: Are you doing the test at the hot spring temperatures? If yes: Do you have checked that the nitrophenol-glucopyranosid is not just hydrolized even without the enzyme at that temperatures in your buffer already? ... it is a quite energy rich bond...)