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Cloning, expression and characterization of human serine racemase mutants
Nováková, Ilona ; Konvalinka, Jan (advisor) ; Brynda, Jiří (referee)
AAbbssttrraacctt Human serine racemase (hSR) is a cytosolic pyridoxal-5'-phosphate dependent enzyme localized in the central nervous system. It synthesizes D-serine, which is an endogenous coagonist for the N-methyl-D-aspartate (NMDA) receptors and plays a key role in excitatory neurotransmission in the brain. Thus, human serine racemase is a promising target for the treatment of neurodegenerative diseases connected with NMDA receptors. However, few specific inhibitors have been identified to date and the crystal structure of hSR has become available only very recently. We decided to perform a random mutagenesis to determine the amino acid residues critical for the enzyme activity. Ser 84 was reported as a catalytic residue along with Lys 56. After analysis of a double mutant S84G/P111L which retained its capability to convert L-serine to pyruvate, we prepared and characterized the single mutant S84G in order to exclude potential effect of the P111L mutation.on the activity of the analyzed enzyme. KKeeyy wwoorrddss:: D-serine; Serine racemase; PLP-dependent enzymes; Random mutagenesis; Racemases
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Analysis of serine racemase expression in the CNS of epileptic patients
Vorlová, Barbora ; Konvalinka, Jan (advisor) ; Maloy Řezáčová, Pavlína (referee)
Serine racemase is a pyridoxal-5'-phosphate dependent enzyme that converts L-serine to D-serine. D-serine is a recognized physiological co-agonist of N-methyl-D-aspartate type of glutamate receptors - key receptors that participate in the neurotransmission in the mammalian brain. Dysfunction of these receptors has been implicated in several neuropathologies, including schizophrenia, brain ischemia, neurodegenerative disorders and epilepsy. Serine racemase is thus a promising pharmaceutical target in these diseases. In this study, three anti-human serine racemase monoclonal antibodies were characterized and the best one was used for the Western blot detection of the enzyme in resected human epileptic tissues. For better interpretation of the results, accuracy of the tissue processing, the protein concentration determination and the Western blot quantification were verified. Finally, the activity of human serine racemase was determined with the L-serine-O-sulfate, the substrate with the highest-affinity to this enzyme. (Thesis in Czech)
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Inhibitors of mouse serine racemase
Vorlová, Barbora
Serine racemase (SR) is a pyridoxal-5'-phosphate-dependent enzyme responsible for biosynthesis of D-serine, a recognized neurotransmitter acting as a co-activator of N-methyl- D-aspartate (NMDA) type of glutamate receptors in the mammalian central nervous system. The hyperfunction of the mentioned receptors have been shown to be implicated in many neuropathological conditions including Alzheimer's disease, amyotrophic lateral sclerosis and epilepsy. To alleviate the symptoms of these diseases, several artificial blockers of NMDA receptors have been introduced into the clinical practice. However, many of these compounds cause undesirable side effects and it is thus necessary to search for either less harmful blockers or regulators of other targets of pharmaceutical intervention that are involved in NMDA receptor activation. In this context, specific inhibition of serine racemase seems to be a promising strategy for regulation of NMDA receptor overstimulation. Mouse serine racemase shares 89% identity with its human ortholog and it was also shown that both enzymes possess similar kinetic parameters and inhibitor specificity. Therefore, the mouse models can be used to search for a potent human serine racemase inhibitor. Although many different compounds for their inhibitory potency towards serine...
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Inhibitors of mouse serine racemase
Vorlová, Barbora
Serine racemase (SR) is a pyridoxal-5'-phosphate-dependent enzyme responsible for biosynthesis of D-serine, a recognized neurotransmitter acting as a co-activator of N-methyl- D-aspartate (NMDA) type of glutamate receptors in the mammalian central nervous system. The hyperfunction of the mentioned receptors have been shown to be implicated in many neuropathological conditions including Alzheimer's disease, amyotrophic lateral sclerosis and epilepsy. To alleviate the symptoms of these diseases, several artificial blockers of NMDA receptors have been introduced into the clinical practice. However, many of these compounds cause undesirable side effects and it is thus necessary to search for either less harmful blockers or regulators of other targets of pharmaceutical intervention that are involved in NMDA receptor activation. In this context, specific inhibition of serine racemase seems to be a promising strategy for regulation of NMDA receptor overstimulation. Mouse serine racemase shares 89% identity with its human ortholog and it was also shown that both enzymes possess similar kinetic parameters and inhibitor specificity. Therefore, the mouse models can be used to search for a potent human serine racemase inhibitor. Although many different compounds for their inhibitory potency towards serine...
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Inhibitors of mouse serine racemase
Vorlová, Barbora ; Konvalinka, Jan (advisor) ; Vaněk, Ondřej (referee)
Serine racemase (SR) is a pyridoxal-5'-phosphate-dependent enzyme responsible for biosynthesis of D-serine, a recognized neurotransmitter acting as a co-activator of N-methyl- D-aspartate (NMDA) type of glutamate receptors in the mammalian central nervous system. The hyperfunction of the mentioned receptors have been shown to be implicated in many neuropathological conditions including Alzheimer's disease, amyotrophic lateral sclerosis and epilepsy. To alleviate the symptoms of these diseases, several artificial blockers of NMDA receptors have been introduced into the clinical practice. However, many of these compounds cause undesirable side effects and it is thus necessary to search for either less harmful blockers or regulators of other targets of pharmaceutical intervention that are involved in NMDA receptor activation. In this context, specific inhibition of serine racemase seems to be a promising strategy for regulation of NMDA receptor overstimulation. Mouse serine racemase shares 89% identity with its human ortholog and it was also shown that both enzymes possess similar kinetic parameters and inhibitor specificity. Therefore, the mouse models can be used to search for a potent human serine racemase inhibitor. Although many different compounds for their inhibitory potency towards serine...
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Cloning, expression and characterization of human serine racemase mutants
Nováková, Ilona ; Brynda, Jiří (referee) ; Konvalinka, Jan (advisor)
AAbbssttrraacctt Human serine racemase (hSR) is a cytosolic pyridoxal-5'-phosphate dependent enzyme localized in the central nervous system. It synthesizes D-serine, which is an endogenous coagonist for the N-methyl-D-aspartate (NMDA) receptors and plays a key role in excitatory neurotransmission in the brain. Thus, human serine racemase is a promising target for the treatment of neurodegenerative diseases connected with NMDA receptors. However, few specific inhibitors have been identified to date and the crystal structure of hSR has become available only very recently. We decided to perform a random mutagenesis to determine the amino acid residues critical for the enzyme activity. Ser 84 was reported as a catalytic residue along with Lys 56. After analysis of a double mutant S84G/P111L which retained its capability to convert L-serine to pyruvate, we prepared and characterized the single mutant S84G in order to exclude potential effect of the P111L mutation.on the activity of the analyzed enzyme. KKeeyy wwoorrddss:: D-serine; Serine racemase; PLP-dependent enzymes; Random mutagenesis; Racemases
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Analysis of serine racemase expression in the CNS of epileptic patients
Vorlová, Barbora ; Maloy Řezáčová, Pavlína (referee) ; Konvalinka, Jan (advisor)
Serine racemase is a pyridoxal-5'-phosphate dependent enzyme that converts L-serine to D-serine. D-serine is a recognized physiological co-agonist of N-methyl-D-aspartate type of glutamate receptors - key receptors that participate in the neurotransmission in the mammalian brain. Dysfunction of these receptors has been implicated in several neuropathologies, including schizophrenia, brain ischemia, neurodegenerative disorders and epilepsy. Serine racemase is thus a promising pharmaceutical target in these diseases. In this study, three anti-human serine racemase monoclonal antibodies were characterized and the best one was used for the Western blot detection of the enzyme in resected human epileptic tissues. For better interpretation of the results, accuracy of the tissue processing, the protein concentration determination and the Western blot quantification were verified. Finally, the activity of human serine racemase was determined with the L-serine-O-sulfate, the substrate with the highest-affinity to this enzyme. (Thesis in Czech)
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