Catching
catalysis in the act: probing enzyme mechanisms using single crystal
microspectrophotometry and X-ray Crystallography
Arwen R. Pearson and Carrie M. Wilmot
Biochemistry, Molecular Biology and Biophysics, The
University of Minnesota, 6-155 Jackson Hall, 321 Church St SE, Minneapolis, MN
55455, USA, (pears079@umn.edu).
Traditionally X-ray crystallography has
provided important insights into the mechanisms of enzyme catalysis. However,
X-ray structures often represent reaction starts, endpoints or kinetic
deadends, and are not direct visualizations of reaction intermediates formed
during turnover. The marriage of spectroscopic techniques and crystallography in
the development of single crystal microspectrophotometers has revolutionised
the field of structural enzymology [1,2].
Many
enzymes have been shown to be catalytically active in the crystalline state
and, if there are no major conformational changes associated with catalysis,
will often remain crystalline during turnover. If an enzyme reaction involves a
chromophore, either as a cofactor or substrate, single crystal
microspectrophotometry can be used to monitor the reaction in the crystal. When
an intermediate of interest is observed to accumulate in the crystal, it can be
flash frozen in a cold nitrogen stream for X-ray structure determination.
I
will present an overview of the commercially available 4DX system [3-5], as
well as methods used in our laboratory to trap reaction intermediates of
methylamine dehydrogenase for structural studies.
1
Hajdu et al. (2000) Nature
Structural Biology 7 1006-1012
2
Wilmot et al. (2002) Met.
Enzymol. 353 301-318
3
Hadfield & Hajdu (1993) J. Appl. Cryst. 26 839-842
4
Sjšgren et al. (2002) J.
Appl. Cryst. 35 113-116
5
http://www.4dx.se/spectrop.htm