Closed model: NMR and X-rays open up enzyme clue

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  • Published: Jul 1, 2017
  • Author: David Bradley
  • Channels: NMR Knowledge Base
thumbnail image: Closed model: NMR and X-rays open up enzyme clue

Closed budget

This is a representation of electron density at the disulphide bond (yellow, between C56 and C163) and in its close vicinity. Credit: Michael Kovermann, University of Konstanz

A structural model of the closed state of the "energy budget" enzyme adenylate kinase emerges from a nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography study by European scientists.

The enzyme adenylate kinase is vital to the management of the energy budget in our cells. The enzyme is a phosphotransferase that catalyses the interconversion of the adenine nucleotides, adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) and continuously monitors phosphate nucleotide levels so that it can maintain cellular homeostasis. To do so it is always changing between an open and a closed state. In the closed form, it is biochemically active and docks the requisite ligand encasing it like a pearl within an oyster.

Now scientists from the Universities of Konstanz, Germany and Umeå University in Sweden, have investigated this closed state in detail to help them construct a structural model at the atomic level of the enzyme docked to its ligand. The structure is now providing them with useful insights into the biochemical mechanisms that govern the cellular energy budget. The team published details of their findings in the journal Proceedings of the National Academy of Sciences (PNAS).

State to state

The opening and closing of adenylate kinase, and thus the capture, processing and release of its ligand takes place some 340 times per second. This is much too rapid to map the individual stages of the process via structural analysis. For structural biologists, gaining information about the closed state of the enzyme at the precise point in the process where biochemical activity peaks is of immense value. Now, NMR expert Michael Kovermann of the University of Konstanz has developed a workaround for this problem. He introduced a disulfide bond as a "chemical thread" of sorts in order to force the enzyme to take on its closed form and trap it in this state. By locking the enzyme in this state the researchers could then take their time to analyse it using NMR spectroscopy and X-ray crystallography without the enzyme cycling through its two states 340 times per second. Kovermann could then, for the first time generate an image of the peak activity.

The closed state work adds to the open structure the team obtained two years ago. "The beauty of it is that, now, we are able to image both liminal states and to make the structural data publicly available," Kovermann explains.

The trapping trick might now be used to investigate other aspects of the enzyme's activity. They have adjusted the disulfide thread and demonstrated that the chemical pull between the enzyme and its ligand increases many times over in the closed state, while productive turnover decreases at the same rate. In other words: the chemical activity of the enzyme is at its highest in the closed state as one might expect, but the turnover decreases because the processed ligand cannot escape the closed shell, which means fewer ligand molecules pass through the enzyme when it is limited in this way.

Enzyme dynamics

Kovermann and his colleagues have now also proven that the structural dynamics of adenylate kinase depend strongly upon the interaction between enzyme and ligand. In other words, upon the presence or absence of the ligand. In order to show this, the team compared the enzyme’s closed state for both variations, with and without a trapped ligand. If there is no ligand, then the closed enzyme's dynamics remains unchanged as compared to its open state. However, once a ligand is present, marked changes are seen. "This behaviour is counterintuitive, it's not what one would expect," explains Kovermann. He adds that it was only thanks to the use of NMR spectroscopy that the team could see this surprising phenomenon; it would be opaque crystallographic studies alone.

Related Links

Proc Natl Acad Sci (USA) 2017, 114, 6298-6303: "Structural basis for ligand binding to an enzyme by a conformational selection pathway"

Article by David Bradley

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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