Optimization of efficiency in the glyoxalase pathway

Abstract

A quantitative kinetic model for the glutathione-dependent conversion of methylglyoxal to D-lactate in mammalian erythrocytes has been formulated, on the basis of the measured or calculated rate and equilibrium constants associated with (a) the hydration of methylglyoxal, (b) the specific base catalyzed formation of glutathione-(R,S)-methylglyoxal thiohemiacetals, (c) the glyoxalase I catalyzed conversion of the diastereotopic thiohemiacetals to (S)-D-lactoylglutathione, and (d) the glyoxalase II catalyzed hydrolysis of (S)-D-lactoylglutathione to form D-lactate and glutathione. The model exhibits the following properties under conditions where substrate concentrations are small in comparison to the Km values for the glyoxalase enzymes: The overall rate of conversion of methylglyoxal to D-lactate is primarily limited by the rate of formation of the diastereotopic thiohemiacetals. The hydration of methylglyoxal is kinetically unimportant, since the apparent rate constant for hydration is (approximately 500-10(3))-fold smaller than that for formation of the thiohemiacetals. The rate of conversion of methylglyoxal to (S)-D-lactoylglutathione is near optimal, on the basis that the apparent rate constant for the glyoxalase I reaction (kcatEt/Km congruent to 4-20 s-1 for pig, rat, and human erythrocytes) is roughly equal to the apparent rate constant for decomposition of the thiohemiacetals to form glutathione and methylglyoxal [k(obsd) = 11 s-1, pH 7]. The capacity of glyoxalase I to use both diastereotopic thiohemiacetals, versus only one of the diastereomers, as substrates represents a 3- to 6-fold advantage in the steady-state rate of conversion of the diastereomers to (S)-D-lactoylglutathione.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Methylglyoxal Metabolism in CHO Cells Grown in Culture

“…Previous studies to model MG metabolism focused on the kinetics of the glyoxalase system (cf. Creighton et al 1988;Martins et al 2001). In this work, a kinetic model that qualitatively describes the distribution of spontaneously reacting MG with proteins was developed and validated in order to better understand MG metabolism in cultured CHO cells.…”

“…This assumption is less than optimal when considering specific amino acid residues. For example, the equilibrium constant for the MG:Na-acetylcysteine interaction (K equil = 5.6 · 10 6 M À1 ; Lo et al 1994) differs significantly from that of MG:reduced glutathione (K equil = 2.8 · 10 5 M À1 ; Creighton et al 1988), a tripeptide containing cysteine. However, if the average equilibrium between MG and the pool of available amino acid residues is considered, this assumption appears reasonable as shown in Figure 3.…”

Arabidopsis Glyoxalase II Contains a Zinc/Iron Binuclear Metal Center That Is Essential for Substrate Binding and Catalysis

“…The increased fluorescence of the C140A mutant enzyme indicates that the environment around the active site tryptophan residues (57 and 198) has been changed by this mutation. It has been suggested previously that substrate binding is the rate-limiting step for glyoxalase II (39). It is possible that the C140A mutation alters the structure of the enzyme to facilitate access of substrate to the active site.…”

Identification and Phenotypic Analysis of Two Glyoxalase II Encoding Genes from Saccharomyces cerevisiae,GLO2 and GLO4, and Intracellular Localization of the Corresponding Proteins

“…It has been shown (43) that the glutathione content in S. cerevisiae varies between different growth conditions. Therefore, another alternative role of the glyoxalase system could be the recycling of glutathione that has been "trapped" spontaneously by methylglyoxal resulting in the formation of hemithioacetal (1,44). This hypothesis is supported by the fact that even in the presence of active glyoxalase I but in the absence of any glyoxalase II enzyme the growth in methylglyoxal-containing medium is inhibited.…”