Observation of covalent intermediates in an enzyme mechanism at atomic resolution

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