Nuclear relaxation studies of the role of the essential metal in glyoxalase I.

Glyoxalase I, a Zn'+-metalloenzyme, induces the slow exchange of the lactoyl Cz proton of the reaction product S-(D-lactoyl)glutathione with the deuterium of 2Hz0, indicating a partial reversal of the enzymatic reaction and confirming a proton transfer mechanism for the isomerization.Paramagnetic effects of the Mn2+substituted enzyme on the longitudinal relaxation rates of the carbon-bound protons of S-(D-lactoyl)glutathione at four frequencies (60, 100, 250, and 360 MHz) yield distances from Mn2+ to these protons ranging from 7.8 to 12.0 A which increase in the order Cys(C,H) a Cys(CBHB) < lactoyl(CH3) -= Cys(CflH~) < Glu(C,Hz) -G1y(C,Hz) -Glu(CpHz).Model building studies indicate that these distances are inconsistent with direct coordination of any of the atoms of S-(D-lactoyl)glutathione by the enzyme-bound Mn2+.The distances do permit an intervening water ligand of the Mn2+ to hydrogen bond to the lactoyl-carbonyl group in a second sphere complex, but are too great for hydrogen bonding to the lactoyl-hydroxyl group.Paramagnetic effects of enzyme-bound MnZ+ on the transverse relaxation rates of the protons of S-(mlactoy1)glutathione as a function of temperature and frequency yield an exchange rate for S-(D-lactoyl)glutathione (21.9 X lo4 s-l) which exceeds V,,) by an order of magnitude indicating that the second sphere product complex detected by NMR dissociates rapidly enough to function in catalysis.The competitive inhibitor S-(pbromobenzy1)glutathione removes essentially all paramagnetic effects of Mnz+glyoxalase I on the relaxation rates of S-(D-1actoyl)glutathione in a manner which is qualitatively and quantitatively consistent with competition at the active site.The presence of an underlying, functional, inner sphere complex is ruled out in two ways.First, from the maximal error in the longitudinal relaxation rates with the Mn2+-enzyme, it is estimated that an inner sphere complex of S-(D-lactoyl)glutathione would dissociate at a rate 5200 s-', which is 1.9 times slower than V,,,.Second, paramagnetic effects of Co2'-substituted glutathione on the relaxation rates of S-(D-lac-