How do enzymes work?

Maybe I am an optimist, but by now, you would think this question would have been answered definitively? I recall as an undergraduate being shown a Cambridge University question set by the famous geneticist and enzymologist JBS Haldane, in which undergraduates were asked to consider the possibilities and challenges surrounding the de novo design of a protein that could fulfill a Biochemical function. I imagine that question was set around 1930! The challenges that remain in delivering this objective are significant, despite the deposition of thousands of protein structures in the PDB and the determination of the sequences of many genomes. In fact the uncertainty that exists in defining gene function remains a significant barrier to the effective "translation" of Molecular Cell Biology into Biotechnological success: the "Holy Grail" of Synthetic Biology.

Perhaps one of the problems that exists is that proteins often have multiple

functions, and we tend to be (naturally) drawn to the earliest description of function. Thus, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, shown right) is well known to Biochemists as the sixth step in Glycolysis, thanks to Embden and Myerhof and Parnas. But it is also implicated in transcriptional regulation, apoptosis and vesicle transport. The point being, that technology (as Sydney Brenner has pointed out) often limits knowledge, and it was possible (over 90 years ago) to identify the GAPDH metabolic reaction, but only 15 years ago to identify its other functions. So GAPDH related sequences in genome analyses are primarily considered glycolysis related. How many other unknown functions of seemingly known genes exist?

This brings me on to the scope of our mutational work on C5 MTases and the forthcoming work from Michael in collaboration with Mark Paine's lab on P450s (and hopefully with Richard Pleass on immune molecules). Since we have a limited (at best) understanding of structure function relationships in protein chemistry, I am interested in using model enzymes to identify those residues that are essential for function (in as unbiased a way as possible). Thereafter, attempt to rationalise these observations using the most appropriate technology and subsequently use this knowledge in a synthetic biology programme. We are at step one at the moment.

The current state of our knowledge in respect of C5 DNA MTases has been

reviewed extensively and therefore we have a good knowledge base. More importantly, by using mcrBC positive and negative strains, we can select for inactivation by mutagenesis. The data from Laila and Sam using our Phusion based error prone PCR system, confirm the essential nature of the catalytic Cys as expected (and this is our best internal control). However, I remain puzzled by the 20 or so point mutations in the TRD of M.HhaI that abolish methylation activity (producing colonies on mcrBC+). I think Laila's subsequent analysis of mutants that are base flipping proficient, but do not complete methyl transfer (as identified by our indirect base flipping screen) are very interesting candidates for structural analysis (we should certainly look carefully at Norbert Reich's NMR data). I shall draw up an infographic to try and simplify the data from Laila's thesis, which are now ready for publication. For me this has demonstrated that we need to accumulate a large data set for M.HhaI. Remember that the mutants that we recover from mcrBC+ plates will be inactive and there is value in establishing which classes of amino acid substitutions inactivate as well as those that do not. I think we should dig in for a comprehensive analysis, since the methodology is now well established. Asma, Michael and Mohammed, you should get together and discuss these concepts: I shall write up Laila's data. Next an update on my take on our historic Pro-Cys Pro-Gly data and the genome analysis that Asma carried out.