Whilst doing kinetics, are your reaction rates linear and positive? I assume you follow your reactions during their measurements (take many data points as a function time). If you follow the disappearance of something, the velocity should be "negative" and hence you need to invert it to get a positive reaction velocity. Only when the reaction rate is positive will you find both Michaelis-Menten parameters to be positive. Also, make sure your reaction rate is faster as [S] increases. If not,  then the protein is either very easily saturated or there is something wrong with your experimental setup.

Also, I would not use Lineweaver-Burk, Hanes-Woolf etc. linear plots to work out kinetic constants, as these introduce high errors in certain plot areas. I suggest you use non-linear regression to determine your Km and Vmax. This can be done in GraphPad Prism, for instance. If you don't have the software, there are a bunch of internet websites where you can input your reaction slopes and determine your parameters. I have used Xuru's NRL tool ( http://www.xuru.org/rt/NLR.asp ) for checking them quickly although I would trust GraphPad more in terms of standard errors etc.

Hope this helps,

Arne

Super helpful thanks!

Arne´s answer is fully correct. I definitely would check the response of the enzyme to changing substrate concetration the way he suggests. In case that your enzyme seems to be saturated at low S concentrations, you could use the integrated Michaleis Menten equation. That allows you to obtain your Km and Vmax in one single assay. If you need help on doing this I´d be glad to help.

So, how d you invert -Vmax to +Vmax? Just doing as  such 1/ -Vmax ?

Best

 Baki

Hello Florencio

Same problem I am facing that when I am plotting Lineweaver-Burk plot, my Km and Vmax is coming negative. Can you suggest me to use the integrated Michaleis Menten equation for calculation of Km and Vmax value for PPO enzyme.