Methylglyoxal synthetase, which catalyzes the formation of methylglyoxal and inorganic phosphate from dihydroxyacetone phosphate, was found in extracts of Proteus vulgaris. An efficient purification procedure utilizing ion exchange column chromatography and isoelectric focusing has been developed. Homogeneity of the enzyme preparation was confirmed by polyacrylamide gel electrophoresis and rechromatography.Two components of methylglyoxal synthetase were obtained upon isoelectric focusing. A comparison of the chemical and physical properties of the two components was carried out. The enzyme is a dimer. In the presence of inorganic phosphate, the hyperbolic saturation kinetics with dihydroxyacetone phosphate are shifted to sigmoidal.

In einer Vielzahl von Arbeiten konnten anti-inflammatorische Eigenschaften von Ethylpyruvat (EP) aufgezeigt werden. An verschiedenen Modellen der Sepsis, des hämorrhagischen Schocks, von Verbrennungsschäden, des Apoplex oder der Ischämie und Reperfusion wurde bei der Behandlung mit EP ein protektiver Effekt sowie eine verminderte Produktion von pro-inflammatorischen Zytokinen nachgewiesen. Als biochemische Grundlage wurde die Interaktion von EP mit dem Transkriptionsfaktor NF-κB identifiziert, die spezifischen Regulationsmechanismen konnten bisher allerdings nicht zufriedenstellend aufgeklärt werden. In dieser Arbeit wurde als eine neue mögliche Erklärung für die anti-inflammatorischen Eigenschaften des EP und weiterer α-oxo-Karbonsäureester die Inhibierung der Glyoxalase I (Glo-I) aufgezeigt. In vitro-Experimente zur Enzymaktivität belegten die Hemmung der Glo-I durch EP, während α-Hydroxy-Karbonsäureester wie L-Ethyllaktat (EL) keine inhibierenden Eigenschaften aufwiesen. Dennoch waren sowohl EP als auch EL und weitere Laktatester in der Lage, die LPS-induzierte Produktion von pro-inflammatorischen Zytokinen wie IL-1β, IL-6, IL-8 und TNF-α von humanen immunkompetenten Zellen zu supprimieren und die Expression von Immunrezeptoren wie HLA-DR, CD14 und CD91 zu modulieren. Somit konnten erstmals anti-inflammatorische Eigenschaften von Laktatestern nachgewiesen sowie eine Verbindung zwischen den Glyoxalase-Enzymen und dem Immunsystem etabliert werden. Diese und weitere Ergebnisse zur Einflussnahme der Karbonsäureester auf die Zellvitalität präsentieren das Glyoxalasesystem als mögliches Ziel neuer Therapiekonzepte für die Immunsuppression und bestätigen dessen Bedeutung für die Entwicklung von Anti-Tumor-Agenzien.

The glyoxalase system, which has been known since 1913, is widely distributed in nature. The system consists of two enzymes, glyoxalase I and glyoxalase II. Methylglyoxal is very unstable and undergoes oxidation and polymerization reactions. One of the purposes of this study was to find a simple, convenient and reproducible method of methylglyoxal preparation. Another objective was the purification of both glyoxalase enzymes employing affinity chromatography as a major step. The purified enzymes were to be characterized by chemical, physical and kinetic properties as an approach to the understanding of the biological function of the system.

No description available.

The glyoxalase pathway is widespread in both prokaryotic and eukaryotic organisms. This system utilizes two enzymes (glyoxalase I (GlxI) and glyoxalase II (GlxII)) to catalyze the formation of D-lactate from the substrates glutathione (GSH) and methylglyoxal (MG). The latter chemical is a harmful byproduct of glycolysis. This thesis gives detailed studies of the behavior of the GlxI enzyme as it pertains to its thiol co-substrate specificity, its structural similarity among its superfamily members (most particularly with the fosfomycin resistance protein (FosA)) and residue identification that would alter its metal selectivity. The thiol co-substrate GSH was thought to be the only thiol utilizied by the glyoxalase system. However, reports identified organisms that utilized the thiols trypanothione (T(SH)2) and glutahionylspermidine (GspdSH) as co-substrates. These organisms, known as the trypanosomes, are very well known in tropical environments to cause diseases. E. coli does not contain T(SH)2 but does contain GspdSH and manufactures the latter in increasing amounts under conditions of cell duress. Substrate specificity studies were conducted replacing GSH with GspdSH and T(SH)2. In addition to this, to ensure the thiols reacted in a true glyoxalase system, substrate specificity studies were also conducted on the second enzyme GlxII and verification of the product D-lactate was performed. To continue, structurally, the enzyme GlxI belongs to the βαβββ superfamily of proteins that are known to have very similar structure but to catalyze very different reactions. Comparing the active site of E. coli GlxI and FosA, there is one significant difference at one residue. Therefore an E56A mutation was performed on GlxI and the mutant bacterium were subjected to growth analysis in the presence of fosfomycin and MG. The mutant enzyme was also tested for its performance in the presence of MG and various divalent metals. Further, the Glx I enzyme from E. coli is known to be active in the presence of non-zinc bivalent metals, while the human counterpart is active in the presence of Zn2+. When one compares GlxI from E. coli with the human GlxI, there are many differences in the primary structure that could be viable areas that determine the metal specificity of the enzyme. Mutation analysis was performed on these areas to determine catalytic performance as well as metal specificity. These studies display how versatile the glyoxalase system is with regard to the use of its thiol co-substrates. These thiols participate in the detoxification pathway for MG in the cell especially under late log phase conditions. Structural studies can give some knowledge concerning the possible evolution of the enzyme among its family members, and is of monumental significance to the scientific community as it relates to enzyme metal selectivity and the development of enzymes over time.

The glyoxalase pathway is widespread in both prokaryotic and eukaryotic organisms. This system utilizes two enzymes (glyoxalase I (GlxI) and glyoxalase II (GlxII)) to catalyze the formation of D-lactate from the substrates glutathione (GSH) and methylglyoxal (MG). The latter chemical is a harmful byproduct of glycolysis. This thesis gives detailed studies of the behavior of the GlxI enzyme as it pertains to its thiol co-substrate specificity, its structural similarity among its superfamily members (most particularly with the fosfomycin resistance protein (FosA)) and residue identification that would alter its metal selectivity. The thiol co-substrate GSH was thought to be the only thiol utilizied by the glyoxalase system. However, reports identified organisms that utilized the thiols trypanothione (T(SH)2) and glutahionylspermidine (GspdSH) as co-substrates. These organisms, known as the trypanosomes, are very well known in tropical environments to cause diseases. E. coli does not contain T(SH)2 but does contain GspdSH and manufactures the latter in increasing amounts under conditions of cell duress. Substrate specificity studies were conducted replacing GSH with GspdSH and T(SH)2. In addition to this, to ensure the thiols reacted in a true glyoxalase system, substrate specificity studies were also conducted on the second enzyme GlxII and verification of the product D-lactate was performed. To continue, structurally, the enzyme GlxI belongs to the βαβββ superfamily of proteins that are known to have very similar structure but to catalyze very different reactions. Comparing the active site of E. coli GlxI and FosA, there is one significant difference at one residue. Therefore an E56A mutation was performed on GlxI and the mutant bacterium were subjected to growth analysis in the presence of fosfomycin and MG. The mutant enzyme was also tested for its performance in the presence of MG and various divalent metals. Further, the Glx I enzyme from E. coli is known to be active in the presence of non-zinc bivalent metals, while the human counterpart is active in the presence of Zn2+. When one compares GlxI from E. coli with the human GlxI, there are many differences in the primary structure that could be viable areas that determine the metal specificity of the enzyme. Mutation analysis was performed on these areas to determine catalytic performance as well as metal specificity. These studies display how versatile the glyoxalase system is with regard to the use of its thiol co-substrates. These thiols participate in the detoxification pathway for MG in the cell especially under late log phase conditions. Structural studies can give some knowledge concerning the possible evolution of the enzyme among its family members, and is of monumental significance to the scientific community as it relates to enzyme metal selectivity and the development of enzymes over time.

In einer Vielzahl von Arbeiten konnten anti-inflammatorische Eigenschaften von Ethylpyruvat (EP) aufgezeigt werden. An verschiedenen Modellen der Sepsis, des hämorrhagischen Schocks, von Verbrennungsschäden, des Apoplex oder der Ischämie und Reperfusion wurde bei der Behandlung mit EP ein protektiver Effekt sowie eine verminderte Produktion von pro-inflammatorischen Zytokinen nachgewiesen. Als biochemische Grundlage wurde die Interaktion von EP mit dem Transkriptionsfaktor NF-κB identifiziert, die spezifischen Regulationsmechanismen konnten bisher allerdings nicht zufriedenstellend aufgeklärt werden. In dieser Arbeit wurde als eine neue mögliche Erklärung für die anti-inflammatorischen Eigenschaften des EP und weiterer α-oxo-Karbonsäureester die Inhibierung der Glyoxalase I (Glo-I) aufgezeigt. In vitro-Experimente zur Enzymaktivität belegten die Hemmung der Glo-I durch EP, während α-Hydroxy-Karbonsäureester wie L-Ethyllaktat (EL) keine inhibierenden Eigenschaften aufwiesen. Dennoch waren sowohl EP als auch EL und weitere Laktatester in der Lage, die LPS-induzierte Produktion von pro-inflammatorischen Zytokinen wie IL-1β, IL-6, IL-8 und TNF-α von humanen immunkompetenten Zellen zu supprimieren und die Expression von Immunrezeptoren wie HLA-DR, CD14 und CD91 zu modulieren. Somit konnten erstmals anti-inflammatorische Eigenschaften von Laktatestern nachgewiesen sowie eine Verbindung zwischen den Glyoxalase-Enzymen und dem Immunsystem etabliert werden. Diese und weitere Ergebnisse zur Einflussnahme der Karbonsäureester auf die Zellvitalität präsentieren das Glyoxalasesystem als mögliches Ziel neuer Therapiekonzepte für die Immunsuppression und bestätigen dessen Bedeutung für die Entwicklung von Anti-Tumor-Agenzien.

The energy metabolism of most tumor cells relies on aerobic glycolysis (Warburg effect) characterized by an increased glycolytic flux that is accompanied by the increased formation of the cytotoxic metabolite methylglyoxal (MGO). Consequently, the rate of detoxification of this reactive glycolytic byproduct needs to be increased in order to prevent deleterious effects to the cells. This is brought about by an increased expression of glyoxalase 1 (GLO1) that is the rate-limiting enzyme of the MGO-detoxifying glyoxalase system. Here, we overexpressed GLO1 in HEK 293 cells and silenced it in MCF-7 cells using shRNA. Tumor-related properties of wild type and transformed cells were compared and key glycolytic enzyme activities assessed. Furthermore, the cells were subjected to hypoxic conditions to analyze the impact on cell proliferation and enzyme activities. Our results demonstrate that knockdown of GLO1 in the cancer cells significantly reduced tumor-associated properties such as migration and proliferation, whereas no functional alterations where found by overexpression of GLO1 in HEK 293 cells. In contrast, hypoxia caused inhibition of cell growth of all cells except of those overexpressing GLO1. Altogether, we conclude that GLO1 on one hand is crucial to maintaining tumor characteristics of malignant cells, and, on the other hand, supports malignant transformation of cells in a hypoxic environment when overexpressed.

The energy metabolism of most tumor cells relies on aerobic glycolysis (Warburg effect) characterized by an increased glycolytic flux that is accompanied by the increased formation of the cytotoxic metabolite methylglyoxal (MGO). Consequently, the rate of detoxification of this reactive glycolytic byproduct needs to be increased in order to prevent deleterious effects to the cells. This is brought about by an increased expression of glyoxalase 1 (GLO1) that is the rate-limiting enzyme of the MGO-detoxifying glyoxalase system. Here, we overexpressed GLO1 in HEK 293 cells and silenced it in MCF-7 cells using shRNA. Tumor-related properties of wild type and transformed cells were compared and key glycolytic enzyme activities assessed. Furthermore, the cells were subjected to hypoxic conditions to analyze the impact on cell proliferation and enzyme activities. Our results demonstrate that knockdown of GLO1 in the cancer cells significantly reduced tumor-associated properties such as migration and proliferation, whereas no functional alterations where found by overexpression of GLO1 in HEK 293 cells. In contrast, hypoxia caused inhibition of cell growth of all cells except of those overexpressing GLO1. Altogether, we conclude that GLO1 on one hand is crucial to maintaining tumor characteristics of malignant cells, and, on the other hand, supports malignant transformation of cells in a hypoxic environment when overexpressed.

No description available.