If the cofactor(NAD) concentration in 1mM and substrate is 10mM in enzyme reaction, the substrate become 9mM after reaction?
and I am using crude enzymes for reaction, can leftovers from cell extract can have cofactors so the reaction can work more? so that substrate can reduce more than 9mM?
Could you rephrase your question. Give a few more details.
What are you measuring? the NAD concentration?
And, until nothing reacts anymore?
What is your assay setup?
???
Achim Recktenwald
I am sending reaction CDCA to 7 keto LCA and 7 keto LCA to UDCA. and latest step is to send CDCA to UDCA. I am using 4 crude enzymes to send the reaction. One of the enzymes is 7 alpha which is used to send CDCA to 7keto LCA. I need to use CDCA , NAD, enzyme and buffer, I already checked its Unit(crude enzyme) as 730unit/mL with the enzyme assay reaction (CDCA 2mM, NAD 0.2mM, enzyme 0.25ug/mL and buffer up to 0.2mL). because I don't know anything about enzyme reaction.. T.T , I want to send the reaction up to CDCA 50mM, I don't know how I set the enzyme unit and cofactor NAD concentration.
You perform an oxidation and a reduction, thus your cofactor will be regenerated. With a crude extract you will most likely have side reactions oxidising your cofactor, thus you would ultimately end up with the oxidised form (keto) of your substance.
Your two alcohols will reach an equilibrium being closo to 1 to 1, thus without withdrawing (esterification for example) you can not reach high yields.
I agree with Anders O Magnusson assuming that NADH and NAD+ are cosubstrates of the two consecutive reactions and there are no interferences with side reactions. You said that your are using 4 crude enzymes.... with these data, it is difficult to predict the final concentrations and the % of conversion. How is the assay? Are you measuring A340 nm to follow the NADH?.
Regards
Francisco Solano if the reaction both uses and generates NADH, how can he monitor this with 340nm?
The resulting signal over the time course should be rather complex, depending on the reaction speeds of the enzymes, their concentration, substrate concentrations, etc.
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