In mammals, the universal sulfuryl group donor molecule 3-phosphoadenosine 5-phosphosulfate (PAPS... more In mammals, the universal sulfuryl group donor molecule 3-phosphoadenosine 5-phosphosulfate (PAPS) is synthesized in two steps by a bifunctional enzyme called PAPS synthetase. The APS kinase domain of PAPS synthetase catalyzes the second step in which APS, the product of the ATP-sulfurylase domain, is phosphorylated on its 3-hydroxyl group to yield PAPS. The substrate APS acts as a strong uncompetitive inhibitor of the APS kinase reaction. We generated truncated and point mutants of the APS kinase domain that are active but devoid of substrate inhibition. Structural analysis of these mutant enzymes reveals the intrasubunit rearrangements that occur upon substrate binding. We also observe intersubunit rearrangements in this dimeric enzyme that result in asymmetry between the two monomers. Our work elucidates the structural elements required for the ability of the substrate APS to inhibit the reaction at micromolar concentrations. Because the ATPsulfurylase domain of PAPS synthetase influences these elements in the APS kinase domain, we propose that this could be a communication mechanism between the two domains of the bifunctional enzyme. Enzyme inhibitors are molecules that bind to enzymes and decrease their activity. Usually, we think of inhibitors as molecules that mimic the substrates, products, or transition state or fit in an allosteric site and, once bound, arrest the enzyme in its catalytic cycle or slow it down. However, even the true substrates (or products) of the reaction can act as inhibitors. In such cases, enzyme kinetic measurements show pronounced
Successive phosphorylation of nucleoside analog prodrugs to their triphosphate forms is required ... more Successive phosphorylation of nucleoside analog prodrugs to their triphosphate forms is required for the pharmacological activity of these compounds in the chemotherapeutic treatment of viral infections and cancer. Human thymidylate kinase (TMPK), apart from its essential physiological role in the biosynthesis of TTP, is also required for the activation of thymidine analogs, such as the clinically used anti-HIV prodrugs AZT and d4T. This enzyme is rate determining in the three-step cascade of AZT phosphorylation. Our structural work on human, yeast and E. coli TMPKs, in conjunction with sequence homology analyses and biochemical data, has demonstrated that three loops are crucial for the function of this enzyme: the first is the highly conserved P-loop motif, which binds and positions the phosphoryl groups of ATP, the second critical loop contains the DR(Y/H) motif that supplies a catalytic arginine and is also important for the binding and positioning of the magnesium ion complexed to ATP, and the third loop is the so-called Lid-region that is a flexible stretch which closes on ATP when it binds. Modifications of the sugar moieties of nucleoside monophosphates are shown to exert drastic effects on the enzyme's conformation and, thus, reduced activity. Our structural work on several TMPKs has formed the basis for generating mutants of human TMPK that are about 100 times more efficient in phosphorylating AZTMP. These enzyme variants could potentially be introduced into HIV-targeted cells in order to significantly improve AZT's antiviral activity.
Biochemical and Biophysical Research Communications, Oct 1, 2003
Modular systems for protein coupling have been applied for anchoring enzyme molecules on liposome... more Modular systems for protein coupling have been applied for anchoring enzyme molecules on liposome surfaces. Two cytoplasmic model enzymes, a-amylase from Escherichia coli (EC. 3.2.1.1) and guanylate kinase from Saccharomyces cerevisiae (EC. 2.7.4.8), were directly coupled by a histidine-tag or indirectly via strep-tag and streptavidin or streptactin linker to a liposome membrane. Though the catalytic properties of the enzymes are generally maintained, stability and specific activity of the enzymes are modified after coupling and are especially influenced by the lipid used for the liposome assembly.
Table S1. Oligonucleotide primers used in this study. Restriction enzyme recognition sites are sh... more Table S1. Oligonucleotide primers used in this study. Restriction enzyme recognition sites are shown in italics (NdeI site for the 5' site, CATATG, and BamHI site at the 3' site, GGATCC).
1979; Elion et al., 1977). This wide substrate specificity gives these kinases their medicinal re... more 1979; Elion et al., 1977). This wide substrate specificity gives these kinases their medicinal relevance. The best studied anticancer gene therapy strategy is the HSV1-TK/ganciclovir system. Suicide gene therapy of cancers and graft-versus-host disease (GVHD) pre
The present study focuses on the formation of microcapsules containing catalytically active L-asp... more The present study focuses on the formation of microcapsules containing catalytically active L-asparaginase (L-ASNase), a protein drug of high value in antileukemic therapy. We make use of the layer-by-layer (LbL) technique to coat protein-loaded calcium carbonate (CaCO 3) particles with two or three poly dextran/poly-L-arginine-based bilayers. To achieve high loading efficiency, the CaCO 3 template was generated by coprecipitation with the enzyme. After assembly of the polymer shell, the CaCO 3 core material was dissolved under mild conditions by dialysis against 20 mM EDTA. Biochemical stability of the encapsulated L-asparaginase was analyzed by treating the capsules with the proteases trypsin and thrombin, which are known to degrade and inactivate the enzyme during leukemia treatment, allowing us to test for resistance against proteolysis by physiologically relevant proteases through measurement of residual L-asparaginase activities. In addition, the thermal stability, the stability at the physiological temperature, and the longterm storage stability of the encapsulated enzyme were investigated. We show that encapsulation of L-asparaginase remarkably improves both proteolytic resistance and thermal inactivation at 37°C, which could considerably prolong the enzyme's in vivo half-life during application in acute lymphoblastic leukemia (ALL). Importantly, the use of low EDTA concentrations for the dissolution of CaCO 3 by dialysis could be a general approach in cases where the activity of sensitive biomacromolecules is inhibited, or even irreversibly damaged, when standard protocols for fabrication of such LbL microcapsules are used. Encapsulated and free enzyme showed similar efficacies in driving leukemic cells to apoptosis.
Herpes virus type 1 thymidine kinase (HSV1-TK) with ganciclovir (GCV) prodrug treatment is the mo... more Herpes virus type 1 thymidine kinase (HSV1-TK) with ganciclovir (GCV) prodrug treatment is the most widely used approach for suicide gene therapy. This ‘suicide’ strategy allows direct reduction of tumors and clearance of donor cells should graft-versus-host disease (GvHD) arise after bone marrow transplantation. Given recent clinical outcomes, this suicide approach may also provide a key safety component for therapeutic gene transfer vectors that integrate. Although suicide gene therapy using HSV1-TK-encoding oncoretroviral vectors has been evaluated in the clinic, the success of this approach has been relatively modest. Reasons for this include: low gene transfer efficacy, reduced expression of the suicide gene, and insufficient conversion of substrate. Our goal is to overcome these limitations by using a novel lentiviral vector (LV) encoding an alternative kinase/prodrug combination. The rational for our innovative suicide gene therapy strategy is two-fold: 1) Lentiviral vectors can efficiently transduce not only dividing cells but also non-dividing cells. 2) Applying a faster viral enzyme like equine herpes virus type 4 thymidine kinase (EHV4-TK) could be advantageous as it has been shown to be kinetically superior to HSV1-TK at GCV phosphorylation. The aim of this study is to evaluate whether LV-mediated gene modification of target cells with EHV4-TK can lead to efficient killing following GCV treatment. We first constructed a LV expression system carrying the wild-type EHV4-TK cDNA with an IRES element followed by a truncated form of human CD19 (hCD19Δ). Human CD19 was chosen as a cell surface marker to allow functional titering of virus and for immuno-enrichment of transduced cells prior to infusion since it is not expressed in the T cell lineage. The truncated form lacks the intracellular domain and therefore does not signal. Use of an IRES element can abrogate some variegated expression seen with vectors having dual promoters. The LV was pseudotyped with VSV-g and concentrated by ultracentrifugation. After one infection, Jurkat cells (human T cell leukemia) showed a more than 80% functional and stable transduction efficiency (MOI = 10). Using hCD19Δ as a selective marker, transduced Jurkat cells were enriched to over 95% positive by immuno-affinity sorting. EHV4-TK-transduced Jurkat cells exhibited increased cell killing in response to GCV treatment (the apoptotic cell indexes with or without GCV were 69.4 ± 1.5 % and 18.8 ± 1.7 %, respectively; n=3). Highly efficient transduction (more than 60%) of primary human T cells was accomplished by a three time exposure to virus over 36 hours at MOI of 20. Next, we found that GCV efficiently killed transduced primary human T cells in a dose dependent manner. We are now comparing the efficiency of GCV conversion by HSV1-TK and EHV4-TK using LV-transduced cells that express the similar protein levels. We are also evaluating intracellular levels of GCV metabolites by HPLC. These results demonstrate that our novel suicide gene therapy strategy has significant potential for many clinical applications.
Screening for genes homologous to adenylate kinase in the yeast Saccharomyces cerevisiae resulted... more Screening for genes homologous to adenylate kinase in the yeast Saccharomyces cerevisiae resulted in the isolation of a homolog of the previously characterized ADK1. The derived protein sequence is most closely related to mammalian GTP:AMP phosphotransferase (adenylate kinase isozyme 3; AK3); this novel gene is therefore named ADK3. Its deletion from the yeast genome does not lead to an observable change in cellular phenotype. A strain defective for both ADK1 and ADK3 is viable. When introduced on a multicopy plasmid into an ADK1-deficient yeast strain, which shows a reduced proliferation rate, ADK3 did not rescue this growth defect. The protein was also highly overexpressed in E. coli cells. However, no change in enzymatic activity was detected in cellular extracts of yeast or bacteria.
Guanylate kinase catalyzes the reversible transfer of the terminal phosphoryl group of ATP to the... more Guanylate kinase catalyzes the reversible transfer of the terminal phosphoryl group of ATP to the acceptor molecule GMP. Detailed analysis of the in vivo function of this enzyme has been limited by the lack of any genetic data. Using oligonucleotides based on amino acid sequence information of the yeast enzyme, the Saccharomyces cerevisiae gene, GUK1, was isolated and characterized. The gene is present in single copy and maps to chromosome IV. Insertional mutagenesis of the GUK1 locus caused recessive lethality, indicating that this enzyme is necessary for vegetative cell growth. Using inducible expression systems, guanylate kinase was produced in large amounts both in S. cerevisiae and in Escherichia coli.
Insights into open/closed conformations of the catalytically active human guanylate kinase as inv... more Insights into open/closed conformations of the catalytically active human guanylate kinase as investigated by small‑angle X‑ray scattering
Functional screenings in droplet-based microfluidics require the analysis of various types of act... more Functional screenings in droplet-based microfluidics require the analysis of various types of activities of individual cells. When screening for enzymatic activities, the link between the enzyme of interest and the information-baring molecule - the DNA - must be maintained to relate phenotypes to genotypes. This linkage is crucial in directed evolution experiments or for the screening of natural diversity. Micro-organisms are classically used to express enzymes from nucleic acid sequences. However, little information is available regarding the most suitable expression system for the sensitive detection of enzymatic activity at the single-cell level in droplet-based microfluidics. Here, we compare three different expression systems for L-Asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1), an enzyme of therapeutic interest that catalyzes the conversion of L-asparagine to L-aspartic acid and ammonia. We developed three expression vectors to produce and localize L-Asparaginase in E. coli either in the cytoplasm, on the surface of the inner membrane (display) or in the periplasm. We show that the periplasmic expression is the most optimal strategy combining both a good yield and a good accessibility for the substrate without the need for lysing the cells. We suggest that periplasmic expression may provide a very efficient platform for screening applications at the single-cell level in microfluidics.
Glutamylcysteine synthetase (GCS) catalyzes the first and ratelimiting step of biosynthesis of a ... more Glutamylcysteine synthetase (GCS) catalyzes the first and ratelimiting step of biosynthesis of a ubiquitous tripeptide glutathione and is a target for development of potential therapeutic agents against parasites and cancer. L-Buthionine-(SR)-sulfoximine (BSO) is a wellknown potent inhibitor of GCS. Clinical trials of BSO have been carried out against alkylating or platinating agent resistance cancers. Crystallographic analyses of GCS-BSO complex will provide an important clue to the catalytic mechanism and structure-assisted drug design for any species of GCSs. The crystal of E. coli GCS in complex with BSO belongs to the space group P2 1 with unit cell constants of a=70.5 Å, b=97.6 Å, c=102.7 Å and =109.5°. The current model was refined to an Rfactor of 21% (R free =24%). g-Phosphate of ATP has already been transferred to the NS sulfoximine nitrogen atom of BSO. We have shown that the cysteine-binding site of the GCS is inductively formed at the binding of cysteine substrate with turn of side chains of Tyr-241 and Tyr-300 to make hydrogen bonds with the carboxyl group of cysteine that w-carboxyl group of BSO mimics. The binding of BSO to the enzyme induces the turn of the side chain of Tyr-241 in spite of the lack of BSO's w-carboxyl group. This conformational change of the side chain may be stabilized by van der Waals interaction between the side chain of Tyr-241 and the glutamate moiety in BSO.
In mammals, the universal sulfuryl group donor molecule 3-phosphoadenosine 5-phosphosulfate (PAPS... more In mammals, the universal sulfuryl group donor molecule 3-phosphoadenosine 5-phosphosulfate (PAPS) is synthesized in two steps by a bifunctional enzyme called PAPS synthetase. The APS kinase domain of PAPS synthetase catalyzes the second step in which APS, the product of the ATP-sulfurylase domain, is phosphorylated on its 3-hydroxyl group to yield PAPS. The substrate APS acts as a strong uncompetitive inhibitor of the APS kinase reaction. We generated truncated and point mutants of the APS kinase domain that are active but devoid of substrate inhibition. Structural analysis of these mutant enzymes reveals the intrasubunit rearrangements that occur upon substrate binding. We also observe intersubunit rearrangements in this dimeric enzyme that result in asymmetry between the two monomers. Our work elucidates the structural elements required for the ability of the substrate APS to inhibit the reaction at micromolar concentrations. Because the ATPsulfurylase domain of PAPS synthetase influences these elements in the APS kinase domain, we propose that this could be a communication mechanism between the two domains of the bifunctional enzyme. Enzyme inhibitors are molecules that bind to enzymes and decrease their activity. Usually, we think of inhibitors as molecules that mimic the substrates, products, or transition state or fit in an allosteric site and, once bound, arrest the enzyme in its catalytic cycle or slow it down. However, even the true substrates (or products) of the reaction can act as inhibitors. In such cases, enzyme kinetic measurements show pronounced
Successive phosphorylation of nucleoside analog prodrugs to their triphosphate forms is required ... more Successive phosphorylation of nucleoside analog prodrugs to their triphosphate forms is required for the pharmacological activity of these compounds in the chemotherapeutic treatment of viral infections and cancer. Human thymidylate kinase (TMPK), apart from its essential physiological role in the biosynthesis of TTP, is also required for the activation of thymidine analogs, such as the clinically used anti-HIV prodrugs AZT and d4T. This enzyme is rate determining in the three-step cascade of AZT phosphorylation. Our structural work on human, yeast and E. coli TMPKs, in conjunction with sequence homology analyses and biochemical data, has demonstrated that three loops are crucial for the function of this enzyme: the first is the highly conserved P-loop motif, which binds and positions the phosphoryl groups of ATP, the second critical loop contains the DR(Y/H) motif that supplies a catalytic arginine and is also important for the binding and positioning of the magnesium ion complexed to ATP, and the third loop is the so-called Lid-region that is a flexible stretch which closes on ATP when it binds. Modifications of the sugar moieties of nucleoside monophosphates are shown to exert drastic effects on the enzyme's conformation and, thus, reduced activity. Our structural work on several TMPKs has formed the basis for generating mutants of human TMPK that are about 100 times more efficient in phosphorylating AZTMP. These enzyme variants could potentially be introduced into HIV-targeted cells in order to significantly improve AZT's antiviral activity.
Biochemical and Biophysical Research Communications, Oct 1, 2003
Modular systems for protein coupling have been applied for anchoring enzyme molecules on liposome... more Modular systems for protein coupling have been applied for anchoring enzyme molecules on liposome surfaces. Two cytoplasmic model enzymes, a-amylase from Escherichia coli (EC. 3.2.1.1) and guanylate kinase from Saccharomyces cerevisiae (EC. 2.7.4.8), were directly coupled by a histidine-tag or indirectly via strep-tag and streptavidin or streptactin linker to a liposome membrane. Though the catalytic properties of the enzymes are generally maintained, stability and specific activity of the enzymes are modified after coupling and are especially influenced by the lipid used for the liposome assembly.
Table S1. Oligonucleotide primers used in this study. Restriction enzyme recognition sites are sh... more Table S1. Oligonucleotide primers used in this study. Restriction enzyme recognition sites are shown in italics (NdeI site for the 5' site, CATATG, and BamHI site at the 3' site, GGATCC).
1979; Elion et al., 1977). This wide substrate specificity gives these kinases their medicinal re... more 1979; Elion et al., 1977). This wide substrate specificity gives these kinases their medicinal relevance. The best studied anticancer gene therapy strategy is the HSV1-TK/ganciclovir system. Suicide gene therapy of cancers and graft-versus-host disease (GVHD) pre
The present study focuses on the formation of microcapsules containing catalytically active L-asp... more The present study focuses on the formation of microcapsules containing catalytically active L-asparaginase (L-ASNase), a protein drug of high value in antileukemic therapy. We make use of the layer-by-layer (LbL) technique to coat protein-loaded calcium carbonate (CaCO 3) particles with two or three poly dextran/poly-L-arginine-based bilayers. To achieve high loading efficiency, the CaCO 3 template was generated by coprecipitation with the enzyme. After assembly of the polymer shell, the CaCO 3 core material was dissolved under mild conditions by dialysis against 20 mM EDTA. Biochemical stability of the encapsulated L-asparaginase was analyzed by treating the capsules with the proteases trypsin and thrombin, which are known to degrade and inactivate the enzyme during leukemia treatment, allowing us to test for resistance against proteolysis by physiologically relevant proteases through measurement of residual L-asparaginase activities. In addition, the thermal stability, the stability at the physiological temperature, and the longterm storage stability of the encapsulated enzyme were investigated. We show that encapsulation of L-asparaginase remarkably improves both proteolytic resistance and thermal inactivation at 37°C, which could considerably prolong the enzyme's in vivo half-life during application in acute lymphoblastic leukemia (ALL). Importantly, the use of low EDTA concentrations for the dissolution of CaCO 3 by dialysis could be a general approach in cases where the activity of sensitive biomacromolecules is inhibited, or even irreversibly damaged, when standard protocols for fabrication of such LbL microcapsules are used. Encapsulated and free enzyme showed similar efficacies in driving leukemic cells to apoptosis.
Herpes virus type 1 thymidine kinase (HSV1-TK) with ganciclovir (GCV) prodrug treatment is the mo... more Herpes virus type 1 thymidine kinase (HSV1-TK) with ganciclovir (GCV) prodrug treatment is the most widely used approach for suicide gene therapy. This ‘suicide’ strategy allows direct reduction of tumors and clearance of donor cells should graft-versus-host disease (GvHD) arise after bone marrow transplantation. Given recent clinical outcomes, this suicide approach may also provide a key safety component for therapeutic gene transfer vectors that integrate. Although suicide gene therapy using HSV1-TK-encoding oncoretroviral vectors has been evaluated in the clinic, the success of this approach has been relatively modest. Reasons for this include: low gene transfer efficacy, reduced expression of the suicide gene, and insufficient conversion of substrate. Our goal is to overcome these limitations by using a novel lentiviral vector (LV) encoding an alternative kinase/prodrug combination. The rational for our innovative suicide gene therapy strategy is two-fold: 1) Lentiviral vectors can efficiently transduce not only dividing cells but also non-dividing cells. 2) Applying a faster viral enzyme like equine herpes virus type 4 thymidine kinase (EHV4-TK) could be advantageous as it has been shown to be kinetically superior to HSV1-TK at GCV phosphorylation. The aim of this study is to evaluate whether LV-mediated gene modification of target cells with EHV4-TK can lead to efficient killing following GCV treatment. We first constructed a LV expression system carrying the wild-type EHV4-TK cDNA with an IRES element followed by a truncated form of human CD19 (hCD19Δ). Human CD19 was chosen as a cell surface marker to allow functional titering of virus and for immuno-enrichment of transduced cells prior to infusion since it is not expressed in the T cell lineage. The truncated form lacks the intracellular domain and therefore does not signal. Use of an IRES element can abrogate some variegated expression seen with vectors having dual promoters. The LV was pseudotyped with VSV-g and concentrated by ultracentrifugation. After one infection, Jurkat cells (human T cell leukemia) showed a more than 80% functional and stable transduction efficiency (MOI = 10). Using hCD19Δ as a selective marker, transduced Jurkat cells were enriched to over 95% positive by immuno-affinity sorting. EHV4-TK-transduced Jurkat cells exhibited increased cell killing in response to GCV treatment (the apoptotic cell indexes with or without GCV were 69.4 ± 1.5 % and 18.8 ± 1.7 %, respectively; n=3). Highly efficient transduction (more than 60%) of primary human T cells was accomplished by a three time exposure to virus over 36 hours at MOI of 20. Next, we found that GCV efficiently killed transduced primary human T cells in a dose dependent manner. We are now comparing the efficiency of GCV conversion by HSV1-TK and EHV4-TK using LV-transduced cells that express the similar protein levels. We are also evaluating intracellular levels of GCV metabolites by HPLC. These results demonstrate that our novel suicide gene therapy strategy has significant potential for many clinical applications.
Screening for genes homologous to adenylate kinase in the yeast Saccharomyces cerevisiae resulted... more Screening for genes homologous to adenylate kinase in the yeast Saccharomyces cerevisiae resulted in the isolation of a homolog of the previously characterized ADK1. The derived protein sequence is most closely related to mammalian GTP:AMP phosphotransferase (adenylate kinase isozyme 3; AK3); this novel gene is therefore named ADK3. Its deletion from the yeast genome does not lead to an observable change in cellular phenotype. A strain defective for both ADK1 and ADK3 is viable. When introduced on a multicopy plasmid into an ADK1-deficient yeast strain, which shows a reduced proliferation rate, ADK3 did not rescue this growth defect. The protein was also highly overexpressed in E. coli cells. However, no change in enzymatic activity was detected in cellular extracts of yeast or bacteria.
Guanylate kinase catalyzes the reversible transfer of the terminal phosphoryl group of ATP to the... more Guanylate kinase catalyzes the reversible transfer of the terminal phosphoryl group of ATP to the acceptor molecule GMP. Detailed analysis of the in vivo function of this enzyme has been limited by the lack of any genetic data. Using oligonucleotides based on amino acid sequence information of the yeast enzyme, the Saccharomyces cerevisiae gene, GUK1, was isolated and characterized. The gene is present in single copy and maps to chromosome IV. Insertional mutagenesis of the GUK1 locus caused recessive lethality, indicating that this enzyme is necessary for vegetative cell growth. Using inducible expression systems, guanylate kinase was produced in large amounts both in S. cerevisiae and in Escherichia coli.
Insights into open/closed conformations of the catalytically active human guanylate kinase as inv... more Insights into open/closed conformations of the catalytically active human guanylate kinase as investigated by small‑angle X‑ray scattering
Functional screenings in droplet-based microfluidics require the analysis of various types of act... more Functional screenings in droplet-based microfluidics require the analysis of various types of activities of individual cells. When screening for enzymatic activities, the link between the enzyme of interest and the information-baring molecule - the DNA - must be maintained to relate phenotypes to genotypes. This linkage is crucial in directed evolution experiments or for the screening of natural diversity. Micro-organisms are classically used to express enzymes from nucleic acid sequences. However, little information is available regarding the most suitable expression system for the sensitive detection of enzymatic activity at the single-cell level in droplet-based microfluidics. Here, we compare three different expression systems for L-Asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1), an enzyme of therapeutic interest that catalyzes the conversion of L-asparagine to L-aspartic acid and ammonia. We developed three expression vectors to produce and localize L-Asparaginase in E. coli either in the cytoplasm, on the surface of the inner membrane (display) or in the periplasm. We show that the periplasmic expression is the most optimal strategy combining both a good yield and a good accessibility for the substrate without the need for lysing the cells. We suggest that periplasmic expression may provide a very efficient platform for screening applications at the single-cell level in microfluidics.
Glutamylcysteine synthetase (GCS) catalyzes the first and ratelimiting step of biosynthesis of a ... more Glutamylcysteine synthetase (GCS) catalyzes the first and ratelimiting step of biosynthesis of a ubiquitous tripeptide glutathione and is a target for development of potential therapeutic agents against parasites and cancer. L-Buthionine-(SR)-sulfoximine (BSO) is a wellknown potent inhibitor of GCS. Clinical trials of BSO have been carried out against alkylating or platinating agent resistance cancers. Crystallographic analyses of GCS-BSO complex will provide an important clue to the catalytic mechanism and structure-assisted drug design for any species of GCSs. The crystal of E. coli GCS in complex with BSO belongs to the space group P2 1 with unit cell constants of a=70.5 Å, b=97.6 Å, c=102.7 Å and =109.5°. The current model was refined to an Rfactor of 21% (R free =24%). g-Phosphate of ATP has already been transferred to the NS sulfoximine nitrogen atom of BSO. We have shown that the cysteine-binding site of the GCS is inductively formed at the binding of cysteine substrate with turn of side chains of Tyr-241 and Tyr-300 to make hydrogen bonds with the carboxyl group of cysteine that w-carboxyl group of BSO mimics. The binding of BSO to the enzyme induces the turn of the side chain of Tyr-241 in spite of the lack of BSO's w-carboxyl group. This conformational change of the side chain may be stabilized by van der Waals interaction between the side chain of Tyr-241 and the glutamate moiety in BSO.
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Papers by Manfred Konrad