13C NMR studies of the product complex of glyoxalase I.

The paramagnetic effects of Mn2+ . glyoxalase I on the 13C relaxation rates of the reaction product, S-(D-lactoyl)glutathione, separately enriched in the lactoyl carbonyl (C-1) and hydroxymethylene (C-2) carbons, have been measured at 62.8 MHz. The 1/fT1p values of C-1 (1100 +/- 120 s-1) and C-2 (712 +/- 290 s-1) and the previously determined tau c (0.74 ns) yield Mn2+ to carbon distances of 5.7 +/- 0.3 and 6.1 +/- 0.5 A, respectively. These distances, together with previously determined Mn2+-proton distances (Sellin, S., Rosevear, P.R., Mannervik, B., and Mildvan, A.S. (1982) J. Biol. Chem. 257, 10023-10029) constrain the thioester carbonyl group of the product to point toward the metal, with the oxygen positioned to accept a hydrogen bond from a water ligand, in a kinetically competent, second sphere complex. Model-building studies indicate that any averaging of multiple second sphere complexes would require as a major contributor at least one conformation with the lactoyl carbonyl oxygen within hydrogen-bonding distance of an intervening water ligand. Such a structure would facilitate polarization of the carbonyl group in the reverse glyoxalase reaction.