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. 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 (1)H 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.
Heine
A
A
Department of Molecular Biology, Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
DeSantis
G
G
Luz
J G
JG
Mitchell
M
M
Wong
C H
CH
Wilson
I A
IA
D000446:Aldehyde-Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution; D001665:Binding Sites; D002384:Catalysis; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003460:Crystallization; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D008239:Lysine / Q000737:chemistry; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011522:Protons; D012267:Ribosemonophosphates / Q000737:chemistry* / Q000378:metabolism*; D012545:Schiff Bases; D014867:Water
D008024:Ligands; D011522:Protons; D012267:Ribosemonophosphates; D012545:Schiff Bases; D014867:Water; C058245:2-deoxyribose 5-phosphate; D000446:Aldehyde-Lyases; C020019:deoxyribose-phosphate aldolase; D008239:Lysine