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

The allosteric transition of threonine-sensitive aspartokinase I-homoserine dehydrogenase I from Escherichia coli has been studied by time-resolved fluorescence spectroscopy. Fluorescence decay can be resolved into 2 distinct classes of tryptophan emitters: a fast component, with a lifetime of about 1.5 ns; and a slow component, with a lifetime of about 4.5 ns. The fluorescence properties of the slow component are modified by the allosteric transition. In the T-form of the enzyme stabilized by threonine, the lifetime of the slow component is longer, with a red-shifted spectrum; its accessibility to quenching by acrylamide becomes slightly higher without any decrease of fluorescence anisotropy. These results indicate a change in polarity of the slow component environment. The quaternary structure change associated with the allosteric transition probably involves global movements of structural domains without leading to any local mobility on the nanosecond time-scale. We suggest that the slow component corresponds to the unique tryptophan of the buried kinase domain.
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PMID:Allosteric transition of aspartokinase I-homoserine dehydrogenase I studied by time-resolved fluorescence. 315 Jun 86

The renaturation of aspartokinase-homoserine dehydrogenase and of some of its smaller fragments has been investigated after complete unfolding by 6 M guanidine hydrochloride. Fluorescence measurements show that a major folding reaction occurs rapidly (in less than a few seconds) after the protein has been transferred to native conditions and results in the shielding of the tryptophan residues from the aqueous solvent; this step also takes place in the fragments and probably corresponds to the independent folding of different segments along the polypeptide chain. The reappearance of the kinase activity, which is an index of the formation of "native" structure within a single chain, is much slower (a few minutes) and has the following properties: it is involved in a kinetic competition with the formation of aggregates; it has an activation energy of 22 +/- 5 kcal/mol; it is not related to a slow reaction in unfolding and thus probably not controlled by the cis-trans isomerization of X-Pro peptide bonds; its rate is inversely proportional to the solvent viscosity. It seems as if this reaction is limited by the mutual arrangement of the regions that have folded rapidly and independently. It is proposed that the mechanism where a fast folding of domains is followed by a slow pairing of folded domains could be generalized to other long chains composed of several domains; such a slow pairing of folded domains would correspond to a rate-limiting process specific to the renaturation of large proteins. The reappearance of the dehydrogenase activity measures the formation of a dimeric species. The dimerization can occur only after each chain has reached its "native" conformation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Mechanism of renaturation of a large protein, aspartokinase-homoserine dehydrogenase. 360 93

In the presence of guanidine hydrochloride concentrations above 2 M, aspartokinase-homoserine dehydrogenase I remains sufficiently soluble so that the fluorescence and circular dichroism of the protein can be measured. Both parameters show that, up to 3 M guanidine hydrochloride, the protein exists in a stable folded state which possesses a large amount of secondary structure and buried tryptophan residues. This intermediate species is probably monomeric; it is reversibly unfolded by guanidine hydrochloride concentrations between 3 and 4 M. This folded species is formed rapidly from unfolded protein when the denaturant is diluted out, and this rapid folding step precedes all the reactivation steps described previously. The existence of a stable monomeric and folded intermediate indicates that the tertiary interactions have a major contribution to the stability of the native structure of aspartokinase-homoserine dehydrogenase I. Similar measurements were performed on two complementary nonoverlapping fragments: a kinase fragment corresponding to the N-terminal third and a dehydrogenase fragment corresponding to the C-terminal two-thirds of the polypeptide chain. Both fragments exist in a stable folded state up to 2.5 M guanidine hydrochloride. Both fragments show cooperative unfolding transitions between 2.5 and 4 M denaturant. The stability of the folded state of a given region is about the same in an isolated fragment and in the entire chain of aspartokinase-homoserine dehydrogenase I: indeed, an equimolar mixture of these two fragments and the intact chain would give about the same results. This indicates that folding of the kinase and dehydrogenase regions occurs independent ly with a single subunit of the entire protein.
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PMID:Folding of aspartokinase-homoserine dehydrogenase I is dominated by tertiary interactions. 637 Mar 3

Recently, target analysis has been re-evaluated as a technique for the determination of molecular sizes (Kempner, E. S. & Schlegel, W. (1979) Anal. Biochem. 92, 2-10). The technique yields the size of the functional unit, i.e. the minimal assembly of structures necessary for a given function such as an enzymatic activity. Using this method, we have not determined the sizes of the functional units for different enzymatic activities on the "arom" conjugate from Euglena, a polyenzyme catalyzing five sequential reactions in the shikimic acid pathway, and on two conjugates from Escherichia coli carrying both aspartokinase and homoserine dehydrogenase activities. In each conjugate, the size for different enzymatic activities was measured and found to be the same. When compared to the molecular weight obtained with other techniques, the target size matched either the entire conjugate (aspartokinase-homoserine dehydrogenase conjugates I and II) or half the unit ("arom" conjugate). The information was obtained with minimal perturbation of the complexes and sparing laborious purification and reconstitution experiments. Tryptophan synthase was irradiated both as an intact conjugate and also as isolated subunits. In both structural forms, beta 2 was identified as the functional unit for the conversion of indole and serine to tryptophan. The results of this study give insight into the structural assembly of these polyenzymes.
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PMID:The functional unit of polyenzymes. Determination by radiation inactivation. 677 Dec 77

As an approach in the study of the evolution of threonine biosynthetic pathways throughout various organisms, the sequences of three enzymes, namely homoserine dehydrogenase, homoserine kinase and threonine synthase, originating from six organisms, namely Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Brevibacterium lactofermentum, Pseudomonas aeruginosa and Saccharomyces cerevisiae, were compared. As a general trend all three enzymatic activities were carried out by proteins sharing sequence relatedness (except for the homoserine kinase of P aeruginosa). Unexpectedly however, for each step one or two enzymes stood out of the main stream: i) for homoserine dehydrogenase, the yeast protein is atypically similar to the E coli enzyme; ii) for homoserine kinase, the P aeruginosa protein shares no similarity with any other species; and iii) for threonine synthase, the B subtilis protein is far distant from the enzymes of other species. Hence in contrast to other biosynthetic pathways such as the tryptophan one, the threonine pathway seems not to have evolved as a whole throughout different organisms but rather each step seems to have been subjected to multiple constraints including substrate-mediated ones and host-specific ones.
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PMID:Evolutionary comparisons of three enzymes of the threonine biosynthetic pathway among several microbial species. 839 99

Fluorescence spectroscopy was used to examine the interaction between human estradiol 17 beta-dehydrogenase (estrogenic 17 beta-hydroxysteroid dehydrogenase, 17 beta-HSD) and the cofactor NADPH. After the binding of NADPH to the enzyme, there was an emission enhancement at 436 nm following an excitation at 295 nm, as compared to the cofactor alone. This phenomenon was attributed to a radiationless transfer of excitation energy from 17 beta-HSD to the enzyme-bound cofactor. The distance of 2.69 nm, between the bound NADPH and the sole tryptophan residue (Trp46) within one subunit, has been determined using fluorescence energy transfer. This result coincides very well with the same distance, recently calculated from the crystallographic coordinates obtained by Ghosh et al. [Ghosh, D., Pletnev, V. Z., Zhu, D.-W., Wawrzak, Z., Duax, W. L., Pangborn, W., Labrie, F. & Lin, S.-X. (1995) Structure 3, 503-513]. Compared to free NADPH, the fluorescence emission of enzyme-bound NADPH was increased in intensity and its maximum blue-shifted from 457 nm to 436 nm. Binding of NADPH to 17 beta-HSD was studied by fluorescence titration. The enzyme binds two molecules of NADPH with a Kd = 0.73 +/- 0.2 microM. The dissociation constant was further confirmed by the method of coenzyme protection against cold inactivation of the enzyme. The binding was little altered in the presence of estradiol-17 beta. The environment of tryptophan residues on the surface of the enzyme is discussed.
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PMID:Fluorescence-energy transfer in human estradiol 17 beta-dehydrogenase-NADPH complex and studies on the coenzyme binding,. 863 27

Rat liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase (3 alpha-HSD) inactivates circulating steroid hormones and is involved in polycyclic aromatic hydrocarbon (PAH) carcinogenesis. It is the only HSD of known structure in the aldo-keto reductase (AKR) superfamily and may provide a paradigm for other mammalian HSDs in this family. The structure of the 3 alpha-HSD.NADP+ binary complex has been determined at 2.7 A resolution and refined to a crystallographic R-factor of 23.4% with good geometry. The model is similar to other binary complexes in the AKR superfamily in that NADP+ binds at the C-terminal end of an alpha/beta barrel. However, it is unique in that NADP+ is bound in two alternate conformations, probably because of the lack of a salt-linked "safety belt" over the pyrophosphate bridge. The structure supports a previously proposed catalytic mechanism for carbonyl reduction in which Tyr 55 is the general acid, and its effective pKa is lowered by the adjacent Lys 84. We present evidence that the structurally distinct short-chain dehydrogenase/reductase (SDR) superfamily may have convergently evolved a similar catalytic mechanism. Insight into substrate binding is offered by a crystal packing contact in which a neighboring molecule inserts a tryptophan residue (Trp 227) into an apolar cleft in 3 alpha-HSD. This cleft is proximal to the bound NADP+ cofactor and contains a surface of apolar residues (Leu 54, Trp 86, Leu 122, Phe 128, Phe 129, Leu 137, Phe 139), making it a likely candidate for the substrate-binding site. Thus, in forming this crystal contact, Trp 227 may mimic a portion of a bound steroid. In addition, we propose that a water molecule in the active site indicates the position of the hydroxyl oxygen in a 3 alpha-hydroxysteroid substrate. Knowledge of the position of this water molecule, combined with the stereochemistry of hydride transfer, suggests that the alpha face of a bound steroid will be oriented toward the side of the apolar cleft containing Trp 86.
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PMID:Structure of 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase complexed with NADP+. 871 59

Rat liver 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD or AKR1C9), a member of the aldo-keto reductase (AKR) superfamily, plays a pivotal role in the inactivation of circulating steroid hormones. It is the most thoroughly characterized HSD of the AKR superfamily and can be used as a template for structure-function studies in other AKR members such as rodent and human 3alpha-, 17beta- and 20alpha-HSDs. Based on the crystal structure of the E.NADP(+) testosterone ternary complex, there are ten residues that line the testosterone binding cavity: T24, L54, Y55, H117, F118, F129, T226, W227, N306 and Y310. Each residue in the cavity, except for the catalytic residues Y55 and H117, was systematically mutated to alanine to determine the role of the individual residues in steroid recognition. Binding data and kinetic parameters (K(d), k(cat), K(m) and k(cat)/K(m)) of the homogeneous mutants were compared with that of the wild type (WT) enzyme. Titration of the intrinsic tryptophan fluorescence with NADPH demonstrated that cofactor binding was unaltered. However, binding of the steroid hormones testosterone and progesterone to the E.NADPH binary complex was affected to varying degrees. The largest effects on K(d) were an 8-fold decrease in affinity for testosterone and a 50-fold decrease in affinity for progesterone. The mutants bound both hormones in the same rank-order except for W227A, where the binding of progesterone was more adversely affected. A series of 3alpha-hydroxysteroid substrates (A/B trans- and cis-ring fused C(19) and C(21) steroids) were used to determine the ability of each mutant to catalyze steroid turnover. The alanine mutants that retained k(cat)/K(m) values similar to WT were those in which alanine substituted short polar residues such as T24A and T226A. The mutants with the lowest catalytic efficiencies were those in which alanine substituted aromatic residues such as W227A and F129A. The loss in catalytic efficiency was due to large changes in k(cat) (up to 1000-fold), but not K(m). Molecular modeling of the alanine mutants showed that changes in the reaction trajectory defined by the angles and distances by groups that participate in catalysis correlate with changes in k(cat). These results highlight the importance of steroid binding site residues in dictating the proper orientation of substrates to achieve high catalytic turnover while having minimal effects on hormone affinity.
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PMID:Steroid-binding site residues dictate optimal substrate positioning in rat 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD or AKR1C9). 1260 26

Candidate attenuators were identified that regulate operons responsible for biosynthesis of branched amino acids, histidine, threonine, tryptophan, and phenylalanine in gamma- and alpha-proteobacteria, and in some cases in low-GC Gram-positive bacteria, Thermotogales and Bacteroidetes/Chlorobi. This allowed us not only to describe the evolutionary dynamics of regulation by attenuation of transcription, but also to annotate a number of hypothetical genes. In particular, orthologs of ygeA of Escherichia coli were assigned the branched chain amino acid racemase function. Three new families of histidine transporters were predicted, orthologs of yuiF and yvsH of Bacillus subtilis, and lysQ of Lactococcus lactis. In Pasteurellales, the single bifunctional aspartate kinase/homoserine dehydrogenase gene thrA was predicted to be regulated not only by threonine and isoleucine, as in E. coli, but also by methionine. In alpha-proteobacteria, the single acetolactate synthase operon ilvIH was predicted to be regulated by branched amino acids-dependent attenuators. Histidine biosynthetic operons his were predicted to be regulated by histidine-dependent attenuators in Bacillus cereus and Clostridium difficile, and by histidine T-boxes in L. lactis and Streptococcus mutans.
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PMID:Attenuation regulation of amino acid biosynthetic operons in proteobacteria: comparative genomics analysis. 1513 44