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Structural Characterization of Enzymes
The proteins in enzyme molecules fold into three-dimensional structures determining how it performs substrate binding, catalysis and regulation. Some of the amino
acids are involved in binding ligands (substrates, intermediates, products, organic
cofactors, metal cofactors or allosteric regulators) and some are actively involved in
catalysis by interacting with the substrate, intermediate or product of the reaction
(Soding et al. 2005). Thus the catalytic activity of enzymes depends on the integrity
of their native protein conformation. The structures of enzymes can be elucidating
by techniques such as spectroscopic methods, x-ray crystallography and more
recently, multidimensional NMR methods.
X-ray Crystallography
X-ray crystallography has been the most explored technique for obtaining threedimensional structures of proteins and in particular enzymes. Knowledge of threedimensional structures is essential to understand reaction mechanisms at the atomic
level (Feiten et al. 2017). One of the pioneers of enzyme crystallography was David
Blow (1931–2004); he shared the Wolf Prize in Chemistry in 1987 for this research
along with David Phillips (1924–1999), who first successfully solved the structure
of chicken egg lysozyme in 1965 (Helliwell 2017). The Wolf Prize 1987 citation
stated “for their contributions to protein X-ray crystallography and to the elucidation of structures of enzymes and their mechanisms of action”. Its structure was
solved to a resolution of 2°A. The diffraction of X-rays caused by a single protein
molecule is too weak to be measured (Rhodes 2006). Therefore, protein crystals are
used for X-ray structure determination to amplify the signal. A protein crystal contains many copies of the molecule neatly arranged in a highly ordered regular three
dimensional array or crystal lattice (Rhodes 2006). The suitability of enzyme crystals for structure determination is based on their ability to interact with X-rays. In
the experimental setup (Fig. 2) a narrow beam of monochromatic X-rays of suitable
wavelength is directed to the crystal which either traverses straight through the crystal, in between the enzyme molecules, or hit the electron clouds of the atoms in the
enzyme molecules. The molecules arranged side-by-side in a periodic way form a
lattice from which the waves diffracted to the same directions accumulate and
strengthen each other to produce diffraction maxima that can be recorded by sensitive detectors (Petsko and Ringe 2004). Enzyme crystals are almost invariably frozen during the X-ray crystallography achieved by directing a cold stream of nitrogen
gas onto the crystal or soaking in a solution called “cryoprotectant” so that, when
frozen, vitrified water, rather than crystalline ice, is formed. Freezing makes the
crystal tolerant to damage by the radiation and usually allows a higher quality and
higher resolution diffraction data, while providing more accurate structural information (Ilari and Savino 2008). Additionally, freezing may sometimes help in
S. A. Rather et al.
Structural Characterization of Enzymes
The proteins in enzyme molecules fold into three-dimensional structures determining how it performs substrate binding, catalysis and regulation. Some of the amino
acids are involved in binding ligands (substrates, intermediates, products, organic
cofactors, metal cofactors or allosteric regulators) and some are actively involved in
catalysis by interacting with the substrate, intermediate or product of the reaction
(Soding et al. 2005). Thus the catalytic activity of enzymes depends on the integrity
of their native protein conformation. The structures of enzymes can be elucidating
by techniques such as spectroscopic methods, x-ray crystallography and more
recently, multidimensional NMR methods.
X-ray Crystallography
X-ray crystallography has been the most explored technique for obtaining threedimensional structures of proteins and in particular enzymes. Knowledge of threedimensional structures is essential to understand reaction mechanisms at the atomic
level (Feiten et al. 2017). One of the pioneers of enzyme crystallography was David
Blow (1931–2004); he shared the Wolf Prize in Chemistry in 1987 for this research
along with David Phillips (1924–1999), who first successfully solved the structure
of chicken egg lysozyme in 1965 (Helliwell 2017). The Wolf Prize 1987 citation
stated “for their contributions to protein X-ray crystallography and to the elucidation of structures of enzymes and their mechanisms of action”. Its structure was
solved to a resolution of 2°A. The diffraction of X-rays caused by a single protein
molecule is too weak to be measured (Rhodes 2006). Therefore, protein crystals are
used for X-ray structure determination to amplify the signal. A protein crystal contains many copies of the molecule neatly arranged in a highly ordered regular three
dimensional array or crystal lattice (Rhodes 2006). The suitability of enzyme crystals for structure determination is based on their ability to interact with X-rays. In
the experimental setup (Fig. 2) a narrow beam of monochromatic X-rays of suitable
wavelength is directed to the crystal which either traverses straight through the crystal, in between the enzyme molecules, or hit the electron clouds of the atoms in the
enzyme molecules. The molecules arranged side-by-side in a periodic way form a
lattice from which the waves diffracted to the same directions accumulate and
strengthen each other to produce diffraction maxima that can be recorded by sensitive detectors (Petsko and Ringe 2004). Enzyme crystals are almost invariably frozen during the X-ray crystallography achieved by directing a cold stream of nitrogen
gas onto the crystal or soaking in a solution called “cryoprotectant” so that, when
frozen, vitrified water, rather than crystalline ice, is formed. Freezing makes the
crystal tolerant to damage by the radiation and usually allows a higher quality and
higher resolution diffraction data, while providing more accurate structural information (Ilari and Savino 2008). Additionally, freezing may sometimes help in
S. A. Rather et al.