Thursday, 25 February 2016

Medical diagnosis with Mutazyme

A cornerstone enzyme for error-prone PCR is Mutazyme, an enzyme with an increase error rate, but less biased than manganese mutagenesis. The manual is very clear and the only major annoyance is that it implicitly says that it makes 1.3 mutations per kb per cycle —that is the log2 of the fold amplification of the target—, whereas it actually makes something around 0.9 mutations per kb per cycle —even with the assumption that no DNA is lost during spin column purification or that DNA cut out of an agarose gel is not shockingly dirty.
However, the biggest mystery is that it says "Not for medical diagnostics".

Monday, 15 February 2016

Biochemical reaction yield and enzyme promiscuity

Reaction yield, i.e. the molar percentage of product over substrate, is often mentioned by chemists, but never by biochemists. My guess is that many enzymes are not perfectly efficient, but have a range of reaction yields.
In The hitchhiker's guide to the galaxy a ship is hidden thanks to the "somebody else's problem" principle, namely people will ignore something problematic that isn't their problem. The reaction yield of enzymes is not something often discussed. The reason is pretty self evident: differentiating between low abundance products would be a minefield of pesky technical issues. So it is somebody else's problem.

Saturday, 6 February 2016

Promiscuously hitchhiking on a pathway

In the seminal 1976 paper on enzyme evolution Roy Jensen first pointed out that the TCA cycle, the ketoadipate route to lysine, pantothenate, isoleucine, valine and leucine biosynthesis all operated via the same mechanistic steps (condensation with an acyl-CoA, rearrangement, oxidation and elimination of a carbon) and conjectured that they descent from a common primordial pathway.
Promiscuity is generally studied with a single enzyme as a model. A few paper tip-toe around it, but I am not sure that there are any that deal specifically with a pathway where the each enzyme along a pathway shows substrate ambiguity towards the promiscuous product of the previous reaction. I mentioned in another post that the branched chain amino acid pathway can produce norvaline, norleucine and homonorleucine when certain enzymes are overexpressed. Each enzyme in the pathway shows substrate ambiguity, so the whole pathway possess substrate ambiguity.
It is not a feature of the oxaloacetate-to-ketoglutarate–like pathways, but can be found in other pathways.