The Origin of the Kinetic Isotope Effect - A Scientific Journey That Took Longer Than Expected

Some scientific journeys last much longer than you expect.

In 2013, I started a collaborative project with Johann Heider on benzylsuccinate synthase—an enzyme that catalyses radical C–C bond formation between toluene and fumarate. In 2015, the paper describing the enzyme’s structure was published, marking the beginning of my molecular dynamics (MD) and QM/MM studies of its reaction mechanism.

Eight years later, we prepared a manuscript combining some of the most advanced enzyme studies I have ever performed: QM/MM modelling of a five-step reaction in two regioselective and two enantiomeric variants, together with two additional radical-quenching mechanisms. All of this was backed up by kinetic measurements, H/D exchange experiments, and kinetic isotope effect measurements using two independent methods. And we did all this for a very large enzyme that is also extremely sensitive to oxygen—meaning that all experiments had to be performed in a glovebox.

We managed to use our model and some rather impressive steady-state equations to predict not only the enzyme’s enantioselectivity, but also the kinetic isotope effect and the mechanism of H/D exchange. We were very proud of the result.

The first journal we submitted to—a high-ranking catalysis journal—rejected the manuscript after five reasonably detailed reviews. However, the reviewers’ comments were sensible, and addressing them substantially improved the paper. We also discovered errors in our equations that had not been spotted by any of the five reviewers. After correcting the manuscript, we submitted it to an even better journal, as the paper had now been significantly improved thanks to the insightful comments.

We were rejected again, but this time the manuscript was transferred to another journal with a decisive suggestion: the paper was too long and should be divided into two parts—a more biochemical part and a more chemical part.

We agreed, despite the fact that this meant moving to a journal with a relatively average impact factor. At that point, we were also quite desperate to finally publish the results of this long-running project.

Both parts were rejected. The second part was rejected on the grounds that the first part had not been published yet. Ironically, the first part had been included in the supplementary material, but the reviewer apparently did not notice it.

We complained to the editors in both cases, as the reviews were, this time, of rather low quality. Fortunately, we were given the opportunity to resubmit. This was followed by two extensive major revisions— yes, two of them for each part.

The recurring criticism was that our methodology was somewhat outdated. And, by that stage, the reviewers had a point: we were already in the ninth and tenth years since the project had started.

However, when one reviewer suggested additional calculations that would have required roughly one million CPU hours of calculations, we got really angry.

Finally, the first part was published, and the second has just arrived—after 28 months under revision.

This is by far the longest time I have ever had a paper under continuous evaluation. In my final letter to the Editor, I wrote frankly that if the manuscript went into yet another round of revision for yet another reviewer, we would withdraw it.

Fortunately, that was not necessary.

Persistence finally paid off.

So, if you are not afraid of some really scary kinetic equations that can explain a great deal about how an enzyme works, dive into:

“The Origin of the Kinetic Isotope Effect and Mechanism of H/D Exchange in Radical C–C Bond Formation Catalyzed by Benzylsuccinate Synthase: Experiments and Microkinetic Modeling”