Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:1.1.1.3 (HSD)
 3,464 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The kinetic mechanisms of the reactions catalyzed by the two catalytic domains of aspartokinase-homoserine dehydrogenase I from Escherichia coli have been determined. Initial velocity, product inhibition, and dead-end inhibition studies of homoserine dehydrogenase are consistent with an ordered addition of NADPH and aspartate beta-semialdehyde followed by an ordered release of homoserine and NADP+. Aspartokinase I catalyzes the phosphorylation of a number of L-aspartic acid analogues and, moreover, can utilize MgdATP as a phosphoryl donor. Because of this broad substrate specificity, alternative substrate diagnostics was used to probe the kinetic mechanism of this enzyme. The kinetic patterns showed two sets of intersecting lines that are indicative of a random mechanism. Incorporation of these results with the data obtained from initial velocity, product inhibition, and dead-end inhibition studies at pH 8.0 are consistent with a random addition of L-aspartic acid and MgATP and an ordered release of MgADP and beta-aspartyl phosphate.
 ...
PMID:The kinetic mechanisms of the bifunctional enzyme aspartokinase-homoserine dehydrogenase I from Escherichia coli. 224 Nov 77

The pH variation of the kinetic parameters was examined for the kinase activity of the bifunctional enzyme aspartokinase--homoserine dehydrogenase I isolated from Escherichia coli. The V/K profile for L-aspartic acid indicates the loss of activity upon protonation of a cationic acid type group with a pK value near neutrality. Incubation of the enzyme with diethyl pyrocarbonate at pH 6.0 results in a loss of enzymic activity. The reversal of this reaction by neutral hydroxylamine, the appearance of a peak at 242 nm for the inactivated enzyme, and the observation of a pK value of 7.0 obtained from variation of the inactivation rate with pH all suggest that enzyme inactivation occurs by modification of histidine residues. The substrate L-aspartic acid protects one residue against inactivation, which implies that this histidine may participate in substrate binding or catalysis. Activity loss was also observed at high pH due to the ionization of a neutral acid group with a pK value of 9.8. The reactions of AK-HSD I with N-acetylimidazole and tetranitromethane have been investigated to obtain information about the functional role of tyrosyl residues in the enzyme. The acylation of tyrosines leads to inactivation of the enzyme, which can then be fully reversed by treatment with hydroxylamine. Incubation of the enzyme with tetranitromethane at pH 9.5 also leads to rapid inactivation, and the substrates of the kinase reaction provide substantial protection against inactivation. However, three tyrosines are protected by substrates, implying a structural role for these amino acids.
 ...
PMID:Aspartokinase-homoserine dehydrogenase I from Escherichia coli: pH and chemical modification studies of the kinase activity. 255 8

Improved purification schemes are reported for the enzymes L-aspartase and aspartokinase-homoserine dehydrogenase I from Escherichia coli. Dye-ligand chromatography on commercially available dye matrices are incorporated as key steps in these purifications. Red A-agarose has a high affinity for L-aspartase, which is then eluted as a homogeneous protein fraction with 1 mM L-aspartic acid. Green A-agarose shows a high binding affinity for the bifunctional enzyme aspartokinase-homoserine dehydrogenase I. Purification is accomplished by elution with NADP+, followed by formation of a ternary complex with NADP and cysteine, a good competitive inhibitor of the homoserine dehydrogenase activity, and rechromatography on Green A-agarose. The final specific activity of each purified enzyme equaled or exceeded previously reported values, the overall yield of enzymes obtained was significantly higher, and these improved purification schemes were found to be more amenable to being scaled up for the production of large quantities of purified enzyme.
 ...
PMID:Purification of aspartase and aspartokinase-homoserine dehydrogenase I from Escherichia coli by dye-ligand chromatography. 389 16

The five enzymes responsible for the conversion of L-aspartate to L-threonine in Escherichia coli were purified to homogeneity and subsequently reconstituted in vitro in ratios approximating those found in vivo. 31P NMR was used to conveniently monitor the rates of consumption of the substrates ATP and NADPH, the accumulation of the intermediates beta-aspartyl phosphate and homoserine phosphate, and the formation of the products ADP, NADP+, and Pi in a single experiment. By this method, the flux of aspartic acid through the enzymes of the pathway was monitored in the absence and in the presence of several alternative substrates and inhibitors. Several known antimetabolites were found to be alternative substrates that ultimately became inhibitors of pathway flux. L-threo-3-Hydroxyaspartic acid was converted to 3-hydroxyhomoserine phosphate by the first four enzymes of the pathway. The antimetabolite L-threo-3-hydroxyhomoserine was found to bind to and inhibit aspartokinase-homoserine dehydrogenase I in a cooperative fashion (I 0.5 = 3 mM, nH = 2.5), similar to the action of the allosteric end product inhibitor L-threonine (I 0.5 = 0.36 mM, nH = 2.4). In the presence of the remaining enzymes of the pathway, however, L-threo-3-hydroxyhomoserine was phosphorylated to the apparent ultimate antimetabolite L-threo-3-hydroxyhomoserine phosphate that was a potent inhibitor of threonine synthase and consequently of L-threonine biosynthesis. When aspartic acid alone was examined as a substrate of the enzymes of the pathway, no accumulation of the beta-aspartyl phosphate and homoserine phosphate intermediates was observed. However, in the presence of either 5 mM L-threo-3-hydroxyhomoserine or 5 mM L-threo-3-hydroxyhomoserine phosphate, homoserine phosphate was found to accumulate. In contrast to the homoserine phosphate and 3-hydroxyhomoserine phosphate intermediates, both of which were very stable, the acylphosphate intermediates beta-aspartyl phosphate and beta-3-hydroxyaspartyl phosphate were highly susceptible to hydrolysis, with first-order rate constants of 4.6 X 10(-3) min-1 and 4.5 X 10(-2) min-1 (pH 7.8, 25 degrees C), respectively.
 ...
PMID:Interaction of aspartate and aspartate-derived antimetabolites with the enzymes of the threonine biosynthetic pathway of Escherichia coli. 615 Sep 34

The coding regions for the Escherichia coli gene for aspartokinase I/homoserine dehydrogenase I (thrA) and the Corynebacterium glutamicum gene for aspartic semialdehyde dehydrogenase (asd) have been subcloned into a Simian Virus 40 (SV40)-based mammalian expression vector. Both enzyme activities are expressed in mouse 3T3 cells after transfer of the corresponding chimaeric gene. The kinetic parameters are similar to those of the native bacterial enzymes, and aspartokinase I/homoserine dehydrogenase I retains its allosteric regulation by threonine. An extract of the cells expressing aspartokinase I/homoserine dehydrogenase I, mixed with one from cells expressing aspartic semialdehyde dehydrogenase, produced homoserine when the mixture was incubated with aspartic acid, ATP and NADPH. The thrA and asd expression cassettes were combined into a single plasmid which, when transfected into 3T3 cells, enabled them to produce homoserine from aspartic acid. Homoserine-producing 3T3 cells were transfected with the plasmid pSVthrB/C (homoserine kinase and threonine synthase) and selected for growth on homoserine. Cell lines isolated from these cells expressed the complete bacterial threonine pathway, were independent of threonine for growth and could be maintained in medium which contained no free threonine. The threonine in the proteins of these cells became enriched in 15N when the culture medium contained [15N]aspartic acid. The production of homoserine and the growth of cells was at a maximum when there was more than 2.5 mM aspartate in the medium. Below this concentration the high Km of aspartokinase limited the flux through the pathway. In the presence of additional aspartic acid the new pathway could sustain a cell cycle time close to that of the same cells cultured in threonine-containing medium.
 ...
PMID:The biosynthesis of threonine by mammalian cells: expression of a complete bacterial biosynthetic pathway in an animal cell. 763 21

11 beta-hydroxysteroid dehydrogenase (11-HSD) catalyzes the interconversion of corticosterone and 11-dehydrocorticosterone in rats, or cortisol and cortisone in humans. The 'liver' or 'Type I' isozyme is a widely distributed glycoprotein that utilizes NADP+ as a co-factor. To study the role of glycosylation in maintaining enzymatic activity, we introduced mutations into the two potential N-linked glycosylation sites (asparagine-X-serine, residues 158-160 and 203-205) predicted from the rat cDNA sequence. Mutagenesis was performed by a PCR based technique, and wild-type (WT) and mutant cDNAs were expressed in Chinese hamster ovary cells after cloning into the pCMV4 vector. At each putative glycosylation site, asparagine (N) was changed to glutamine (Q) or aspartic acid (D), and serine (S) changed to alanine (A). All three modifications of the first site (N158Q, N158D, S160A) had minimal (75-100% of WT) effects on dehydrogenase activity and caused a mild (50-75% of WT) decrease in reductase activity. In contrast, mutations at the second site had marked effects, with N203Q and N203D completely abolishing both dehydrogenase and reductase activities and S205A decreasing both activities to about 20% of WT. The double mutation of S160A and S205A also abolished all activity, even though the enzyme carrying each mutation alone was, at least, partially active. The results suggest that N203 (which is highly but not completely conserved in short chain dehydrogenase enzymes) is essential for activity of 11-HSD. N-linked glycosylation may be necessary for full activity or stability of the enzyme.
 ...
PMID:Mutations in putative glycosylation sites of rat 11 beta-hydroxysteroid dehydrogenase affect enzymatic activity. 771 Oct 58

The objectives of this study were to determine whether stress attenuates the pituitary LH response to excitatory amino acids by altering expression of glutamate receptor 1 (GluR1) and N-methyl-D-aspartic acid (NMDA) receptor mRNA levels in the hypothalamus or pituitary, and assess whether stress influences testicular levels of mRNA or protein for steroidogenic enzymes. Three hours (h) of immobilization stress was associated with a greater than 7-fold increase in serum corticosterone, and a marked reduction in serum testosterone (T) concentrations. Stress did not significantly alter hypothalamic or pituitary GluR1 and NMDA receptor mRNA levels. Although transcript levels for P450SCC and P45017alpha mRNA in the testis were unchanged in stressed rats, western blotting of testicular fractions revealed reduced amounts of P450SCC and 3beta-HSD, but not P45017alpha. The data suggest that immobilization stress reduces T production by suppressing the translation of transcripts for P450SCC and 3beta-HSD, but the attenuated LH response of stressed animals to NMDA is not mediated by altered hypothalamic or pituitary expression of GluR1 and NMDA receptor levels.
 ...
PMID:Expression of mRNA and proteins for testicular steroidogenic enzymes and brain and pituitary mRNA for glutamate receptors in rats exposed to immobilization stress. 1062 2

Fungal homoserine dehydrogenase (HSD) is required for the biosynthesis of threonine, isoleucine and methionine from aspartic acid, and is a target for antifungal agents. HSD from the yeast Saccharomyces cerevisiae was overproduced in Escherichia coli and 25 mg of soluble dimeric enzyme was purified per liter of cell culture in two steps. HSD efficiently reduces aspartate semialdehyde to homoserine (Hse) using either NADH or NADPH with kcat/Km in the order of 10(6-7) M(-1) x s(-1) at pH 7.5. The rate constant of the reverse direction (Hse oxidation) was also significant at pH 9.0 (kcat/Km approximately 10(4-5) M(-1) x s(-1)) but was minimal at pH 7.5. Chemical modification of HSD with diethyl pyrocarbonate (DEPC) resulted in a loss of activity that could be obviated by the presence of substrates. UV difference spectra revealed an increase in absorbance at 240 nm for DEPC-modified HSD consistent with the modification of two histidines (His) per subunit. Amino acid sequence alignment of HSD illustrated the conservation of two His residues among HSDs. These residues, His79 and His309, were substituted to alanine (Ala) using site directed mutagenesis. HSD H79A had similar steady state kinetics to wild type, while kcat/Km for HSD H309A decreased by almost two orders of magnitude. The recent determination of the X-ray structure of HSD revealed that His309 is located at the dimer interface [B. DeLaBarre, P.R. Thompson, G.D. Wright, A.M. Berghuis, Nat. Struct. Biol. 7 (2000) 238-244]. The His309Ala mutant enzyme was found in very high molecular weight complexes rather than the expected dimer by analytical gel filtration chromatography analysis. Thus the invariant His309 plays a structural rather than catalytic role in these enzymes.
 ...
PMID:Characterization of yeast homoserine dehydrogenase, an antifungal target: the invariant histidine 309 is important for enzyme integrity. 1134 14

Three genes from the aminoethoxyvinylglycine (AVG)-producing Streptomyces sp. NRRL 5331 involved in threonine biosynthesis, hom, thrB and thrC, encoding homoserine dehydrogenase (HDH), homoserine kinase (HK) and threonine synthase (TS), respectively, have been cloned and sequenced. The hom and thrC genes appear to be organized in a bicistronic operon as deduced by disruption experiments. The thrB gene, however, is transcribed as a monocistronic transcript. The encoded proteins are quite similar to the HDH, HK and TS proteins from other bacterial species. The overall organization of these three genes, in the order hom-thrC-thrB, differs from that in other bacteria and is similar to that reported in the Streptomyces coelicolor genome sequence. This is the first time in which the gene cluster for the three last steps of threonine biosynthesis has been characterized from a streptomycete. Disruption of thrC indicated that threonine is not a direct precursor for AVG biosynthesis in Streptomyces sp. NRRL 5331 and suggested that the branching point of the aspartic acid-derived biosynthetic route of this metabolite should lie earlier on the threonine biosynthetic route.
 ...
PMID:Characterization of the hom-thrC-thrB cluster in aminoethoxyvinylglycine-producing Streptomyces sp. NRRL 5331. 1198 15

FKBP12 is a conserved member of the prolyl-isomerase enzyme family and serves as the intracellular receptor for FK506 that mediates immunosuppression in mammals and antimicrobial actions in fungi. To investigate the cellular functions of FKBP12 in Saccharomyces cerevisiae, we employed a high-throughput assay to identify mutations that are synthetically lethal with a mutation in the FPR1 gene, which encodes FKBP12. This screen identified a mutation in the HOM6 gene, which encodes homoserine dehydrogenase, the enzyme catalyzing the last step in conversion of aspartic acid into homoserine, the common precursor in threonine and methionine synthesis. Lethality of fpr1 hom6 double mutants was suppressed by null mutations in HOM3 or HOM2, encoding aspartokinase and aspartate beta-semialdehyde dehydrogenase, respectively, supporting the hypothesis that fpr1 hom6 double mutants are inviable because of toxic accumulation of aspartate beta-semialdehyde, the substrate of homoserine dehydrogenase. Our findings also indicate that mutation or inhibition of FKBP12 dysregulates the homoserine synthetic pathway by perturbing aspartokinase feedback inhibition by threonine. Because this pathway is conserved in fungi but not in mammals, our findings suggest a facile route to synergistic antifungal drug development via concomitant inhibition of FKBP12 and Hom6.
 ...
PMID:FKBP12 controls aspartate pathway flux in Saccharomyces cerevisiae to prevent toxic intermediate accumulation. 1547 Feb 57


1 2 Next >>