[hpl912]

in reactions catalyzed by alpha-kgd complex, it gave me results which shows activity (all about 0.03 dA/min at 340nm).
I've run 3 additional reactions where the first was without substrate NAD+ but replaced with NADP+, the second without CoA and the third without TPP in the reaction mixture. All of these results in no activity (very close to 0 dA/min).

1) when replaced with NADP+ that result no activity means it can't generate NADPH? both substrates acts as removing protons but why it doesn't work with NADP+? maybe my experiment went wrong? or it is specific only to NAD+?
If it's the additional phosphate group that makes it inactive, then it means FAD will work and generate FADH as NAD?

2) when no CoA or TPP was in the mixture, there was no observed activity neither. so does this means alpha-kgd needs decarboxylation for it to work (CoA and TPP are decarboxylators and activators here if i understood correctly)?

thanks in advance for any information

[Yggdrasil]

1) when replaced with NADP+ that result no activity means it can't generate NADPH? both substrates acts as removing protons but why it doesn't work with NADP+? maybe my experiment went wrong? or it is specific only to NAD+?
If it's the additional phosphate group that makes it inactive, then it means FAD will work and generate FADH as NAD?

Enzyme active sites are often very selective and substrates (such as NAD+) are often described as fitting into the enzyme active site like a hand in a glove or like a key in a lock.  NADP+ and NAD+ are almost identical except for one phosphate residue.  However, enzyme active sites can distinguish between the two forms.  This can be explained in the fact that NAD+/NADH and NADP+/NADPH play different roles in the cell.  The cell maintains a high NAD+/NADH ratio to use NAD+ as an oxidizing agent to store energy from oxidation reactions in catabolism.  On the other hand, cells maintain a low NADP+/NADPH ratio in order to use NADPH as a reducing agent in various anabolic reactions.  Because cells use there different molecules for different purposes, enzymes have evolved to discriminate between the two molecules very effectively.

2) when no CoA or TPP was in the mixture, there was no observed activity neither. so does this means alpha-kgd needs decarboxylation for it to work (CoA and TPP are decarboxylators and activators here if i understood correctly)?

That's a fairly good interpretation of the results.