01 January 2019 5 8K Report

I’m working with a rather complicated ping pong, bi-bi double displacement reductase enzyme that oxidizes NADH and then reduces a substrate. Interestingly, the enzyme has intrinsic NADH oxidase activity in the absence of the substrate. Of course, upon addition of substrate, the velocity of the reaction increases and NADH is consumed more rapidly. The intrinsic activity introduces a baseline velocity and is complicating our kinetic analyses a bit. Complicating it even further is a noted double substrate inhibition pattern (apparently not uncommon with ping-pong mechanism enzymes). We’ve run a full course of analyses, varying the NADH as well as the substrate and we're now trying to fit the data to an appropriate equation. My question is: How do I take the ‘intrinsic activity’ of the enzyme into account (if I need to at all)? Can anyone recommend an equation and appropriate software that can do this? We currently have SigmaPlot, GraphPad, and Enzfitter (for the double substrate inhibition model). Thanks in advance for any guidance, ideas, and/or discussion.

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