Fluorescence and nuclear relaxation enhancement studies of the binding of glutathione derivatives to manganese-reconstituted glyoxalase I from human erythrocytes. A model for the catalytic mechanism of the enzyme involving a hydrated metal ion.

The apoenzyme of glyoxalase I (EC 4.4.1.5) from human erythrocytes was prepared by removal of Zn2+ with ethylenediaminetetraacetic acid (EDTA). Methanol was used as a stabilizing agent. Extended dialysis was required to remove EDTA from the resulting solution of apoenzyme. Reconstitution with Mn2+ was followed by measuring enzyme activity, electron paramagnetic resonance of free Mn2+ ions, and nuclear magnetic resonance of water protons. The holoenzyme contained two Mn2+ per protein dimer and had approximately 50% of the catalytic activity of the native enzyme. The binding of the cosubstrate glutathione (gamma-L-glutamyl-L-cysteinylglycine), the product S-D-lactoyl-glutathione, and the competitive inhibitor S-(p-bromo-benzyl)glutathione was monitored by the quenching of the intrinsic tryptophan fluorescence and by the proton relaxation enhancement of water bound to Mn2+ in the active site of the enzyme. The dissociation constants were 1.1 mM, 0.42 mM, and 0.54 microM for glutathione, S-D-lactoylglutathione, and S-(p-bromobenzyl)glutathione, respectively. The temperature and frequency dependences of the longitudinal and transverse paramagnetic relaxation rates, 1/T1p and 1/T2p, were studied for water. The results were analyzed in terms of correlation and exchange times. In addition proton and deuteron relaxation rates were measured in parallel at two different magnetic fields. Good agreement between the two approaches of analysis was noticed. The data show that two water molecules are bound in the first coordination sphere of Mn2+ in the active site of glyoxalase I. When S-(p-bromobenzyl)glutathione or S-D-lactoylglutathione is bound to the enzyme, only one exchangeable water molecule could be detected, indicating occlusion of the second water molecule. An enediol mechanism involving the metal-bound water is proposed for the catalysis effected by glyoxalase I.