Observation of Covalent Intermediates in an Enzyme Mechanism at Atomic Resolution
Andreas Heine,1*
Grace DeSantis,2*
John G. Luz,1
Michael Mitchell,2
Chi-Huey Wong,23
Ian A. Wilson13
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.
Present address: Diversa Corporation, 4955 Directors
Place, San Diego, CA 92121, USA.
To whom correspondence should be addressed. E-mail:
wong{at}scripps.edu (C.-H.W.); wilson{at}scripps.edu (I.A.W.)
