A hydrogen bond: What did it do for them? Part 3

We turn now to the remaining article cited in support of the assertion that a neutral-neutral hydrogen bond will contribute no more that 1.5kcal/mol to binding affinity. We have not found the evidence (see parts 1 and 2) presented so far in support of this claim to be overly convincing so we're desperately hoping that this article will clear the rather muddy waters. The article was in fact the first one to be cited and pre-dates the other two. For those of our loyal readers who care about such trivia, the journal has a high impact factor. Why have we left it until last to review this article? We have our reasons which may become apparent to some of you.

Before we get stuck into the business at hand, let's take a quick look at what we've learned so far. First, if you're going to claim that you've established an upper limit for the contribution of a hydrogen bond you do need to demonstrate that your hydrogen bonds are optimal in terms of geometry and solvent exposure. We also learned that you can't really take the contributions of one type of acceptor (e.g amide oxygen or hydroxyl) and extrapolate them to other types of acceptor (e.g. aromatic nitrogen). Lastly, the contribution of a hydrogen bond may well depend on the number of other intermolecular hydrogen bonds between ligand and protein.

Delighted that you've taken all this in because it's time to take a look at the featured article. This is a well known, heavily-cited publication that describes the use of protein engineering to analyse hydrogen bonding and biological specificity. The enzyme is tyrosyl-tRNA synthetase and we should point out at the outset that it's a nice paper.



The starting point for the analysis is a a crystal structure (see fig 1 in the article) of tyrosyl adenylate (see above) bound to the enzyme. Amino acids whose side chains are observed to form hydrogen bonds with the ligand are systematically modified (site-directed mutagenesis) and the effects of these mutations are quantified by comparing kcat/Km values with that for the wild type enzyme. There are 11 hydrogen bonds between ligand and protein. Five of these can be counted as having a charged partner either in the ligand or in the protein and five can be regarded as true neutral-neutral hydrogen bonds. Just in case you though we were losing it, the eleventh hydrogen bond, that between GLN195 and carbonyl oxygen. Although this looks like a neutral-neutral hydrogen bond it isn't really. That carbonyl oxygen is one of the carboxylate oxygen atoms in the E.Tyr complex and it is no surprise to learn that this is the most important hydrogen bond for stabilising the transition state.

Two of the five neutral-neutral hydrogen bond involve backbone atoms, leaving three that can be probed by conventional mutagnesis. These involve the side chains of CYS35, THR51 and TYR34 and none appears to contribute more than 1.18kcal/mol. THR51 and TYR34 both deploy hydroxyl groups and you've already heard our concerns about interpreting contributions of hydroxyl groups. Interestingly it is the thiol of CYS35that appears to make the largest contribution despite thiols being weaker hydrogen bonbd acceptors than hydroxyls.

So there you have it. We have contributions of three neutral-neutral hydrogen bonds. How likely do you think it is that one of these represents the upper limit for a neutral-neutral hydrogen bond?

We have now reviewed the evidence presented by the defence in support the assertion that a neutral-neutral hydrogen bond will contribute no more than 1.5kcal/mol. We hope that you have enjoyed the journey or at least found it to be a character building process. In the next Crapshoot we will pass judgement. Will it be 10 hours of community service or 10 minutes of Old Sparky?

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