Note special date.
Single molecule fluorescence spectroscopy has evolved into a powerful technique for monitoring conformational changes of enzymes. Despite the power of the single molecule approach to also investigate the kinetics of the catalytic reaction itself, the field of single enzyme kinetics is only about to leave the proof-of-principle stage. Using so-called fluorogenic substrates S, converted by the enzyme E into the fluorescent product P, the sequence of individual enzymatic turnovers can be followed using a confocal fluorescence microscope [1]. This sequence contains the complete kinetic information and serves as the starting point for the construction of kinetic schemes.
After introducing the detection principle, I will discuss the importance of the substrate design [2,3] and the data analysis procedure [4] for obtaining accurate turnover sequences. I will further give an example how this approach can be used for studying the pH-dependent activity of the enzyme alpha-chymotrypsin and highlight drawbacks and future directions. Once technological limitations have been overcome, single turnover detection has the potential to resolve detailed kinetic schemes of enzymes as a function of environmental influences, mutations and regulation events.
[1] V. I. Claessen, H. Engelkamp, P. C. M Christianen, J. C. Maan, R. J. M. Nolte, K. Blank and A. E. Rowan. (2010) Single-Biomolecule Kinetics: The Art of Studying a Single Enzyme. Annu Rev Anal Chem 3:319
[2] T. G. Terentyeva, W. Van Rossom, M. Van der Auweraer, K. Blank and J. Hofkens (2011) Morpholinecarbonyl-Rhodamine 110 based Substrates for the Determination of Protease Activity with Accurate Kinetic Parameters. Bioconjugate Chem 22:1932
[3] T. G. Terentyeva, J. Hofkens, T. Komatsuzaki, K. Blank and C.-B. Li (2013) Time-resolved fluorescence spectroscopy of an -chymotrypsin catalyzed reaction. J Phys Chem B submitted
[4] T. G. Terentyeva, H. Engelkamp, T. Komatsuzaki, A. E. Rowan, J. Hofkens, C.-B. Li and K. Blank (2012) Dynamic Disorder in Single Enzyme Experiments: Facts and Artifacts. ACS Nano 6:346