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Science 12 October 2001:
Vol. 294. no. 5541, pp. 369 - 374
DOI: 10.1126/science.1063601
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Reports

Observation of Covalent Intermediates in an Enzyme Mechanism at Atomic Resolution

Andreas Heine,1* Grace DeSantis,2*dagger John G. Luz,1 Michael Mitchell,2 Chi-Huey Wong,23ddagger Ian A. Wilson13ddagger

In classical enzymology, intermediates and transition states in a catalytic mechanism are usually inferred from a series of biochemical experiments. Here, we derive an enzyme mechanism from true atomic-resolution x-ray structures of reaction intermediates. Two ultra-high resolution structures of wild-type and mutant D-2-deoxyribose-5-phosphate (DRP) aldolase complexes with DRP at 1.05 and 1.10 angstroms unambiguously identify the postulated covalent carbinolamine and Schiff base intermediates in the aldolase mechanism. In combination with site-directed mutagenesis and 1H nuclear magnetic resonance, we can now propose how the heretofore elusive C-2 proton abstraction step and the overall stereochemical course are accomplished. A proton relay system appears to activate a conserved active-site water that functions as the critical mediator for proton transfer.

1 Department of Molecular Biology,
2 Department of Chemistry,
3 Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
*   These authors contributed equally to this work.

dagger    Present address: Diversa Corporation, 4955 Directors Place, San Diego, CA 92121, USA.

ddagger    To whom correspondence should be addressed. E-mail: wong{at}scripps.edu (C.-H.W.); wilson{at}scripps.edu (I.A.W.)

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