1H NMR detection of immobilized water molecules within a strong distal hydrogen-bonding network of substrate-bound human heme oxygenase-1

Solution 1H NMR is used to probe the environments of the donor protons of eight strong hydrogen bonds on the distal side of the heme substrate in the cyanide-inhibited, substrate-bound complex of human heme oxygenase, hHO. It is demonstrated that significant magnetization transfer from the bulk water signal to the eight labile protons does not result from chemical exchange, but from direct nuclear Overhauser effect due to the dipolar interaction of these labile protons with "ordered" water molecules. The enzyme labile proton to water proton distances are estimated at approximately 3 A. It is proposed that the role of the strong hydrogen-bonding network is to immobilize numerous water molecules which both stabilize the activated hydroperoxy species and funnel protons to the active site.     

Enzyme-catalyzed synthesis of furanosyl nucleotides

A bacterial alpha-d-glucopyranosyl-1-phosphate thymidylyltransferase was found to couple four hexofuranosyl-1-phosphates, as well as a pentofuranosyl-1-phosphate, with deoxythymidine 5'-triphosphate, providing access to furanosyl nucleotides. The enzymatic reaction mixtures were analyzed by electrospray ionization mass spectrometry and NMR spectroscopy to determine the anomeric stereochemistry of furanosyl nucleotide products. This is the first demonstration of a nucleotidylyltransferase discriminating between diastereomeric mixtures of sugar-1-phosphates to produce stereopure, biologically relevant furanosyl nucleotides.