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Title
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Direct structural observation of an acyl-enzyme intermediate in the hydrolysis of an ester substrate by elastase.
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Authors
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X.Ding,
B.F.Rasmussen,
G.A.Petsko,
D.Ringe.
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Ref.
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Biochemistry, 1994,
33,
9285-9293.
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PubMed id
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Abstract
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The method of X-ray crystallographic cryoenzymology has been used to determine
the crystal structure of a kinetically significant species on the reaction
pathway of a crystalline enzyme. The structure of a specific acyl-enzyme
intermediate in the elastase-catalyzed hydrolysis of the
N-carbobenzoxy-L-alanine p-nitrophenyl ester has been determined and refined
against X-ray diffraction data at 2.3-A resolution. The difference Fourier
electron density map clearly shows electron density for the trapped acyl-enzyme.
The acyl-enzyme was formed at -26 degrees C and was stabilized at -55 degrees C
during data collection, taking advantage of the glass transition in protein
dynamics that occurs at around -50 degrees C.
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