Kinetic analysis of the human erythrocyte glyoxalase system using 1H NMR and a computer model.
1H NMR was used with methylglyoxal, purified by an HPLC technique, to study the kinetics of the human erythrocyte glyoxalase system. 1H NMR enabled the direct measurement of the time-dependent changes in concentrations of the two hydrates of methylglyoxal, which have not previously been directly measurable, as well as measurement of substrates and products of the glyoxalase enzyme system in the human red blood cell. A computer model of the reaction scheme was developed and NMR data numerically analyzed, thus allowing a complete kinetic description of the reactions. The rate constants describing the chemical equilibria between the hydrated species of methylglyoxal were determined by this numerical analysis or by a saturation-transfer technique, and found to be much slower (by several orders of magnitude) than previously determined by other methods. The kinetic parameters describing the enzyme-catalyzed reactions were also determined from experiments using a dilute haemolysate that was added to solutions of methylglyoxal and reduced glutathione (GSH). The maximal velocity of glyoxalase 1 is threefold greater (Vmax = 70.4 +/- 4.7 mmol.min-1.1 packed cells-1) than glyoxalase 2(Vmax = 24 +/- 5 mmol.min-1.1 packed cells-1) and it exhibits threefold-greater affinity for its substrate (Km = 0.46 +/- 0.04 mM) than the second enzyme (Km = 1.5 +/- 0.4 mM). Both enzymes are subject to competitive inhibition; glyoxalase 1 by reduced glutathione (KiGSH = 7.88 +/- 0.16 mM) and glyoxalase 2 by the hemithioacetal (HTA) of methylglyoxal and GSH (KiHTA = 0.29 +/- 0.04 mM).
Rae
C
C
Department of Biochemistry, University of Sydney, Australia.
Berners-Price
S J
SJ
Bulliman
B T
BT
Kuchel
P W
PW
D004912:Erythrocytes / Q000201:enzymology* / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D007791:Lactoylglutathione Lyase / Q000097:blood*; D009682:Magnetic Resonance Spectroscopy; D011765:Pyruvaldehyde / Q000378:metabolism; D013869:Thiolester Hydrolases / Q000097:blood*; D000099301:Hydroxyacylglutathione Hydrolase
D007791:Lactoylglutathione Lyase; D011765:Pyruvaldehyde; D013869:Thiolester Hydrolases; D000099301:Hydroxyacylglutathione Hydrolase