Human erythrocytes glucose-6-phosphate dehydrogenase: Labelling of a reactive lysyl residue by pyridoxal-5′-phosphate

Reaction of glucose-6-phosphate dehydrogenase from human erythrocytes with pyridoxal-5′-phosphate causes 80% loss of activity. The substrate glucose-6-phosphate fully protects the enzyme against this inhibition, which is reversible upon dilution, but becomes irreversible after treatment with NaBH₄. We presume that pyridoxal-5′-phosphate forms with the enzyme a Schiff base which is reduced by NaBH₄. One mole of N-?-pyridoxyl-lysine is formed per mole of enzyme subunit when the remaining activity reaches its minimal level of 20%.     

Chemical modification of an essential lysine at the active site of enoyl-CoA reductase in fatty acid synthetase

Fatty acid synthetase from goose uropygial gland was inactivated by treatment with pyridoxal 5′-phosphate. Malonyl-CoA and acetyl-CoA did not protect the enzyme whereas NADPH provided about 70% protection against this inactivation. 2′-Monophospho-ADP-ribose was nearly as effective as NADPH while 2′-AMP, 5′-AMP, ADP-ribose, and NADH were ineffective suggesting that pyridoxal 5′-phosphate modified a group that interacts with the 5′-pyrophosphoryl group of NADPH and that the 2′-phosphate is necessary for the binding of the coenzyme to the enzyme. Of the seven component activities catalyzed by fatty acid synthetase only the enoyl-CoA reductase activity was inhibited. Inactivation of both the overall activity and enoyl-CoA reductase of fatty acid synthetase by this compound was reversed by dialysis or dilution but not after reduction with NaBH₄. The modified protein showed a characteristic Schiff base absorption (maximum at 425 nm) that disappeared on reduction with NaBH₄ resulting in a new absorption spectrum with a maximum at 325 nm. After reduction the protein showed a fluorescence spectrum with a maximum at 394 nm. Reduction of pyridoxal phosphate-treated protein with NaB³H₄ resulted in incorporation of ³H into the protein and paper chromatography of the acid hydrolysate of the modified protein showed only one fluorescent spot which was labeled and ninhydrin positive and had an R_f identical to that of authentic N⁶-pyridoxyllysine. When [4-³H]pyridoxal phosphate was used all of the ³H, incorporated into the protein, was found in pyridoxyllysine. All of these results strongly suggest that pyridoxal phosphate inhibited fatty acid synthetase by forming a Schiff base with the ?-amino group of lysine in the enoyl-CoA reductase domain of the enzyme. The number of lysine residues modified was estimated with [4-³H]pyridoxal-5′-phosphate/NaBH₄ and by pyridoxal-5′-phosphate/NaB³H₄. Scatchard analysis showed that modification of two lysine residues per subunit resulted in complete inactivation of the overall activity and enoyl-CoA reductase of fatty acid synthetase. NADPH prevented the inactivation of the enzyme by protecting one of these two lysine residues from modification. The present results are consistent with the hypothesis that each subunit of the enzyme contains an enoyl-CoA reductase domain in which a lysine residue, at or near the active site, interacts with NADPH.     

Crystal structure of glutamate-1-semialdehyde aminotransferase from Bacillus subtilis with bound pyridoxamine-5'-phosphate

Glutamate-1-semialdehyde aminotransferase (GSA-AT), also named glutamate-1-semialdehyde aminomutase (GSAM), a pyridoxamine-5′-phosphate (PMP)/pyridoxal-5′-phosphate (PLP) dependent enzyme, catalyses the transamination of the substrate glutamate-1-semialdehyde (GSA) to the product 5-Aminolevulinic acid (ALA) by an unusual intramolecular exchange of amino and oxo groups within the catalytic intermediate 4,5-diaminovalerate (DAVA). This paper presents the crystal structure of GSA-AT from Bacillus subtilis (GSA-AT_Bsu) in its PMP-bound form at 2.3 Å resolution. The structure was determined by molecular replacement using the Synechococcus GSAM (GSAM_Syn) structure as a search model. Unlike the previous reported GSAM/GSA-AT structures, GSA-AT_Bsu is a symmetric homodimer in the PMP-bound form, which shows the structural symmetry at the gating loop region with open state, as well as identical cofactor (PMP) binding in each monomer. This observation of PMP in combination with an “open” lid supports one characteristic feature for this enzyme, as the catalyzed reaction is believed to be initiated by PMP. Furthermore, the symmetry of GSA-AT_Bsu structure challenges the previously proposed negative cooperativity between monomers of this enzyme.     

Covalent modification of the solubilized rat liver vitamin K-dependent carboxylase with pyridoxal-5′-phosphate

The carboxylation of the pentapeptide substrate, Phe-Leu-Glu-Glu-Ile, by a rat microsomal vitamin K-dependent carboxylase was stimulated two- to threefold at pyridoxal-5′-P concentrations between 0.5 and 1.0 mm. This stimulation was reduced at concentrations higher than 1.0 mm. The K_m for the pentapeptide was lowered twofold in the presence of 1 mm pyridoxal-5′-P. The activation by pyridoxal-5′-P is specific, as 1 mm pyridoxal, pyridoxine, pyridoxine-5′-P, pyridoxamine, pyridoxamine-5′-P, or 4-pyridoxic acid did not stimulate the pentapeptide carboxylation rate. All six analogs, as well as formaldehyde and acetaldehyde, inhibited the carboxylation reaction in a concentration-dependent manner. The activation of the carboxylase by pyridoxal-5′-P appeared to be mediated by its direct binding to the enzyme via Schiff base formation. Sodium borohydride reduction of solubilized microsomes in the presence of pyridoxal-5′-P, followed by dialysis to remove unbound material, resulted in a carboxylase preparation with a specific activity twice that of the untreated control microsomes. The derivatized enzyme was not further stimulated by added pyridoxal-5′-P. This derivatized carboxylase could be obtained in the absence of pentapeptide and divalent cations. The stimulation of the carboxylase activity by divalent cations and pyridoxal-5′-P was mediated at separate site(s) on the enzyme. Studies of the NH₂-terminal pyridoxalated pentapeptide with both a normal and PLP-modified enzyme, in the presence and absence of PLP, demonstrated competition of the pentapeptide PLP moiety to a PLP site on the enzyme. It was concluded that pyridoxal-5′-P forms a covalent attachment to an ?-NH₂ of a lysine near the active site of the carboxylase.     

Inhibition by pyridoxal-5'-phosphate of gamma-aminobutyric acid receptor binding to synaptic membranes of cat cerebellum.

The binding of $\text{[}{}^3\text{H]γ-aminobutyric}$ acid to cat cerebellar membranes is $\text{reversibly}$ inhibited in a competitive manner by pyridoxal-5′-phosphate present during the binding assay. Structural analogues of the inhibitor have no such effect. If, on the other hand, the membranes are preincubated with pyridoxal-5′-phosphate followed by the addition of sodium borohydride, a rapid, $\text{irreversible}$ inhibition of subsequent γ-aminobutyric acid binding is observed. Since pyridoxal-5′-phosphate is known to inactivate certain enzymes by reacting with essential lysine residues, the present results suggest that such a lysine residue may be present within the γ-aminobutyric acid receptor.     

Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides. Interaction of the enzyme with coenzymes and coenzyme analogs.

Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides utilizes either NAD⁺ or NADP⁺ as coenzyme. Kinetic studies showed that NAD⁺ and NADP⁺ interact with different enzyme forms (Olive, C., Geroch, M. E., and Levy, H. R. (1971) J. Biol. Chem.246, 2047–2057). In the present study the techniques of fluorescence quenching and fluorescence enhancement were used to investigate the interaction between Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase and coenzymes. In addition, kinetic studies were performed to examine interaction between the enzyme and various coenzyme analogs. The maximum quenching of protein fluorescence is 5% for NADP⁺ and 50% for NAD⁺. The dissociation constant for NADP⁺, determined from fluorescence quenching measurements, is 3 μm, which is similar to the previously determined K_m of 5.7 μm and K_i of 5 μm. The dissociation constant for NAD⁺ is 2.5 mm, which is 24 times larger than the previously determined K_m of 0.106 mm. Glucose 1-phosphate, a substrate-competitive inhibitor, lowers the dissociation constant and maximum fluorescence quenching for NAD⁺ but not for NADP⁺. This suggests that glucose 6-phosphate may act similarly and thus play a role in enabling the enzyme to utilize NAD⁺ under physiological conditions. When NADPH binds to the enzyme its fluorescence is enhanced 2.3-fold. The enzyme was titrated with NADPH in the absence and presence of NAD⁺; binding of these two coenzymes is competitive. The dissociation constant for NADPH from these measurements is 24 μm; the previously determined K_i is 37.6 μm. The dissociation constant for NAD′ is 2.8 mm, in satisfactory agreement with the value obtained from protein fluorescence quenching measurements. Various compounds which resemble either the adenosine or the nicotinamide portion of the coenzyme structure are coenzyme-competitive inhibitors; 2′,5′-ADP, the most inhibitory analog tested, gives NADP⁺-competitive and NAD⁺-noncompetitive inhibition, consistent with the kinetic mechanism previously proposed. By using pairs of coenzyme-competitive inhibitors it was shown in kinetic studies that the two portions of the NAD⁺ structure cannot be accommodated on the enzyme simultaneously unies they are covalently linked. Fluorescence studies showed that there are both “buried” and “exposed” tryptophan residues in the enzyme structure.     

Active-site modification of glycerol-3-phosphate dehydrogenase by pyridoxal-5′-phosphate

Glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) from rabbit skeletal muscle is inhibited by pyridoxal-5′-phosphate. The inhibition observed in steady-state kinetic studies is competitive with respect to dihydroxyacetone phosphate and uncompetitive with respect to NADH. Similar inhibition was found for a series of related compounds which in order of increasing effectiveness of inhibition were: 4-deoxypyridoxine < pyridoxal < pyridoxic acid < pyridoxal-5′-phosphate < pyridoxine and pyridoxamine-5′-phosphate. Pyridoxal-5′-phosphate also reacts slowly with the enzyme to produce an adduct which upon treatment with sodium borohydride results in irreversible modification of the enzyme. The nature of the adduct was investigated by titration of the enzyme with pyridoxal-5′-phosphate, uv-visible and fluorescence spectroscopy, amino acid analysis, and peptide mapping. All such studies are consistent with a single, highly reactive lysyl residue on each enzyme subunit. Protection of the lysyl residue against modification was afforded by the presence of NADH. The modified enzyme, on the other hand, possessed kinetic properties similar to the native enzyme including a nearly identical inhibition constant for pyridoxal-5′-phosphate. Pyridoxal-5′-phosphate, therefore, seems to have two sites of interaction on the enzyme: a reversible binding site competitive with substrate and a Schiff-base site protected by NADH. These properties of glycerol-3-phosphate dehydrogenase set it apart from functionally similar enzymes.     

Synthesis and structure–activity relationships of carboxylic acid derivatives of pyridoxal as P2X receptor antagonists

Carboxylic acid derivatives of pyridoxal were developed as potent P2X₁ and P2X₃ receptor antagonists with modifications of a lead compound, pyridoxal-5′-phosphate-6-azophenyl-2′,5′-disulfonate (5b, iso-PPADS). The designing strategies included the modifications of aldehyde, phosphate or sulfonate groups of 5b, which may be interacted with lysine residues of the receptor binding pocket, to weak anionic carboxylic acid groups. The corresponding carboxylic acid analogs of pyridoxal-5′-phosphate (1), 13 and 14, showed parallel antagonistic potencies. Also, most of 6-azophenyl derivatives (24–28) of compound 13 or 14 showed potent antagonistic activities similar to that of 5b at human P2X₃ receptors with 100 nM range of IC₅₀ values in two-electrode voltage clamp (TEVC) assay system on the Xenopus oocyte. The results indicated that aldehyde and phosphoric or sulfonic acids in 5b could be changed to a carboxylic acid without affecting antagonistic potency at mouse P2X₁ and human P2X₃ receptors.     

Balance between Metabolite Accumulation and Transport in Relation to Photosynthesis by Isolated Spinach Chloroplasts

The relationship between the rate of orthophosphate (Pi) transport into the stroma and the rate of CO₂ fixation by intact chloroplasts was investigated. High Pi concentrations in the medium lead to a depletion of stromal metabolites, due to excessive Pi transport into the stroma, resulting in the inhibition of CO₂ fixation. This inhibitory effect of Pi is released by inhibitors of Pi transport, such as pyrophosphate, citrate or pyridoxal-5-phosphate. The latter compound appeared to be specially valuable in inhibiting Pi transport without affecting stromal reactions.     

Serine Hydroxymethyltransferase from Mung Bean (Vigna radiata) Is Not a Pyridoxal-5'-Phosphate-Dependent Enzyme

Serine hydroxymethyltransferase from mammalian and bacterial sources is a pyridoxal-5′-phosphate-containing enzyme, but the requirement of pyridoxal-5′-phosphate for the activity of the enzyme from plant sources is not clear. The specific activity of serine hydroxymethyltransferase isolated from mung bean (Vigna radiata) seedlings in the presence and absence of pyridoxal-5′-phosphate was comparable at every step of the purification procedure. The mung bean enzyme did not show the characteristic visible absorbance spectrum of a pyridoxal-5′-phosphate protein. Unlike the enzymes from sheep, monkey, and human liver, which were converted to the apoenzyme upon treatment with l-cysteine and dialysis, the mung bean enzyme similarly treated was fully active. Additional evidence in support of the suggestion that pyridoxal-5′-phosphate may not be required for the mung bean enzyme was the observation that pencillamine, a well-known inhibitor of pyridoxal-5′-phosphate enzymes, did not perturb the enzyme spectrum or inhibit the activity of mung bean serine hydroxymethyltransferase. The sheep liver enzyme upon interaction with O-amino-d-serine gave a fluorescence spectrum with an emission maximum at 455 nm when excited at 360 nm. A 100-fold higher concentration of mung bean enzyme-O-amino-d-serine complex did not yield a fluorescence spectrum. The following observations suggest that pyridoxal-5′-phosphate normally present as a coenzyme in serine hydroxymethyltransferase was probably replaced in mung bean serine hydroxymethyltransferase by a covalently bound carbonyl group: (a) inhibition by phenylhydrazine and hydroxylamine, which could not be reversed by dialysis and or addition of pyridoxal-5′ phosphate; (b) irreversible inactivation by sodium borohydride; (c) a spectrum characteristic of a phenylhydrazone upon interaction with phenylhydrazine; and (d) the covalent labeling of the enzyme with substrate/product serine and glycine upon reduction with sodium borohydride. These results indicate that in mung bean serine hydroxymethyltransferase, a covalently bound carbonyl group has probably replaced the pyridoxal-5′-phosphate that is present in the mammalian and bacterial enzymes.     

Exploration of inhibitors for diaminopimelate aminotransferase

Bacteria and higher plants make l-lysine from diaminopimelic acid (DAP). In mammals l-lysine is an essential amino acid that must be acquired from the diet as the biosynthetic pathway is absent for this key constituent of proteins. Recently, ll-diaminopimelate aminotransferase (ll-DAP-AT), a pyridoxal-5′-phosphate (PLP)-dependent enzyme, was reported to catalyze a key step in the route to l-lysine in plants and Chlamydia. Specific inhibitors of this enzyme could thus potentially serve as herbicides or antibiotics that are non-toxic to mammals. In this work, 29,201 inhibitors were screened against ll-DAP-AT and the IC₅₀ values were determined for the top 46 compounds. An aryl hydrazide and rhodanine derivatives were further modified to generate 20 analogues that were also tested against ll-DAP-AT. These analogues provide additional structure–activity relationships (SAR) that are useful in guiding further design of inhibitors.     

Purification and characterization of l-allo-threonine aldolase from Aeromonas jandaei DK-39

l-allo-Threonine aldolase (l-allo-threonine acetaldehyde-lyase), which exhibited specificity for l-allo-threonine but not for l-threonine, was purified from a cell-free extract of Aeromonas jandaei DK-39. The purified enzyme catalyzed the aldol cleavage reaction of l-allo-threonine (K_m=1.45 mM, V_max=45.2 μmol min⁻¹ mg⁻¹). The activity of the enzyme was inhibited by carbonyl reagents, which suggests that pyridoxal-5′-phosphate participates in the enzymatic reaction. The enzyme does not act on either l-serine or l-threonine, and thus it can be distinguished from serine hydroxy-methyltransferase (l-serine:tetrahydrofolate 5,10-hydroxy-methyltransferase, EC 2.1.2.1) or l-threonine aldolase (EC 4.1.2.5).     

Aminooxy analog of histamine is an efficient inhibitor of mammalian l-histidine decarboxylase: combined in silico and experimental evidence

Histamine plays highlighted roles in the development of many common, emergent and rare diseases. In mammals, histamine is formed by decarboxylation of l-histidine, which is catalyzed by pyridoxal-5′-phosphate (PLP) dependent histidine decarboxylase (HDC, EC 4.1.1.22). The limited availability and stability of the protein have delayed the characterization of its structure–function relationships. Our previous knowledge on mammalian HDC, derived from both in silico and experimental approaches, indicates that an effective competitive inhibitor should be capable to form an “external aldimine-like structure” and have an imidazole group, or its proper mimetic, which provides additional affinity of PLP-inhibitor adduct to the HDC active center. This is confirmed using HEK-293 cells transfected to express human HDC and the aminooxy analog of histidine, 4(5)-aminooxymethylimidazole (O-IMHA, IC₅₀ ≈ 2 × 10^?7 M) capable to form a PLP–inhibitor complex (oxime) in the enzyme active center. Taking advantage of the availability of the human HDC X-ray structure, we have also determined the potential interactions that could stabilize this oxime in the active site of mammalian HDC.     

S1 nuclease of Aspergillus oryzae: characterization of the associated phosphomonoesterase activity

S₁ nuclease (EC 3.1.30.1) of Aspergillus oryzae was found to catalyze the hydrolysis of 2′- or 3′-phosphomonoester groups from several mono- and oligonucleotides. The specificity of the enzyme for mononucleotide substrates was determined by steady-state kinetic measurements at pH 4.5. The values of V were similar for all ribonucleoside 3′-phosphates tested, and they were 50–400 times greater than those for the corresponding deoxyribonucleotides or ribonucleoside 2′-phosphates. Purine nucleotides had lower apparent K_m values than pyrimidine nucleotides. Apparent K_m values of mononucleotides were also strongly dependent on the type of sugar and the positions of phosphoryl groups. Substrate specificity, as expressed by $\text{V}\text{K}{}_{\mathrm{m}}$, occurred in the following order: ribonucleoside 3′,5′-bisphosphate > ribonucleoside 3′-phosphate > deoxyribonucleoside 3′,5'-bisphosphate > deoxyribonucleoside 3′-phosphate ≈ ribonucleoside 2′-phosphate. S₁ nuclease also catalyzed the dephosphorylation of the dinucleotide ApAp at a high rate and the release of PP_i from adenosine 3′-diphosphate 5′-phosphate at a low rate. The phosphomonoesterase activity of the enzyme was competitively inhibited by single-stranded DNA and 5′-nucleotides. Apparent K_i values for adenosine compounds occurred in the order ATP < ADP < AMP ? adenosine. Tests of S₁ nuclease for phosphotransferase activity at pH 4.5 and 7.0 were negative.     

Interaction of pyridoxal phosphate analogues with apoenzymes of gamma-cystathionase and serine sulphhydrase

Comparative studies have been done of the interactions of some coenzyme analogues with the apoenzymes of γ-cystathionase (EC 4.2.1.15) from rat liver and serine sulphhydratase (EC 4.2.1.22) from chicken liver — pyridoxal-phosphate-dependent enzymes catalysing reactions of H₂S release from L-cystein $\text{via}$ α,β-elimination and β-substitution, respectively. It was found that minor modifications (substitutions) in the structure of pyridoxal-5′-phosphate (pyridoxal-P; PLP) result in marked lowering of affinity of the analogues for the apoenzymes. Considerable differences were observed between the various apoenzymes in regard to the mode of their interaction with the pyridoxal-P analogues used.     

Stimulation of vitamin K-dependent carboxylation by pyridoxal-5'-phosphate.

This paper presents evidence that the approximately two-fold increase in vitamin K-dependent carboxylation of the pentapeptide PheLeuGluGluLeu, but not of endogenous protein substrate, brought about by pyridoxal-5′-phosphate, is due to binding of the pyridoxal-5′-phosphate to microsomal enzyme(s), rather than to the pentapeptide. Pyridoxine inhibits this peptide carboxylation, while pyridoxal, pyridoxamine, and pyridoxamine-5′-phosphate have no effect on the reaction.     

The C-S Lyases of Higher Plants : Direct Comparison of the Physical Properties of Homogeneous Alliin Lyase of Garlic (Allium sativum) and Onion (Allium cepa)

Garlic and onion alliin lyases, although from closely related species, have many differences. The two enzymes differ in their K_m values, pH optima, and isoelectric points. There is a major difference in their molecular weight and subunit structure. The garlic holoenzyme has a molecular weight of 85,000 and consists of two subunits of molecular weight 42,000. The onion enzyme has a holoenzyme molecular weight of 200,000 composed of four subunits of molecular weight 50,000. The onion enzyme is much more difficult to dissociate into its subunits which suggests differences in subunit interaction between the two enzymes. The dimeric stucture of the garlic and the tetrameric structure of the onion enzyme is consistent with a coenzyme content (pyridoxal-5′-phosphate) equivalent to one mole per subunit. The two enzymes vary vastly in their spectra, the onion enzyme having a lower pyridoxal-5′-phosphate absorbance at 430 nanomoles and an inability to react with l-cysteine. Both enzymes are glycoproteins and bind to concanavalin A-Sepharose columns. The onion alliin lyase binds more tightly than the garlic enzyme. The amino acid content of both enzymes is similar as is the carbohydrate content. However, upon hydrolysis the onion lyase does yield more mannose units than the garlic enzyme which is consistent with the former's stronger affinity for concanavalin A.     

Inhibition of human pyridoxal kinase by 2-acetyl-4-((1R,2S,3R)-1,2,3,4-tetrahydroxybutyl)imidazole (THI)

Abstract

2-Acetyl-4-((1R,2S,3R)-1,2,3,4-tetrahydroxybutyl)imidazole (THI) is observed as a minor contaminant in caramel food colourings (E?150c). Feeding experiments with rodents have revealed a significant lymphopenic effect that has been linked to the presence of THI in these food colourings. Pyridoxal kinase inhibition by THI has been suggested, but not demonstrated, as a mode of action as it leads to lowered levels of pyridoxal-5′-phosphate, which are known to cause lymphopenia. Recently, THI was also shown to inhibit sphingosine-1-phosphate lyase causing comparable immunosuppressive effects and derivatives of THI are being developed for the treatment of rheumatoid arthritis in humans. Interestingly, sphingosine-1-phosphate lyase activity depends on pyridoxal-5′-phosphate, which in turn is provided by pyridoxal kinase. This report shows that THI does inhibit pyridoxal kinase with competitive and mixed-type non-competitive behaviour towards its two substrates, pyridoxal and ATP, respectively. The corresponding inhibition constants are in the low millimolar range.     

Estriol and estradiol interactions with the estrogen receptor in vivo and in vitro

The cytosolic estrogen receptor (calf uterus) bound to estradiol (E₂) at 0°C changes from a state with fast into a state with slow E₂ dissociation rates when placed at 28°C. This temperature accelerated transition in receptor affinity for its ligand takes place within 10 min at 28°C. Similarly, receptor bound to estriol (E₃) at 0°C changes, when heated, from a state with fast into a state with slow E₃ dissociation. The main difference between RE₂ and RE₃ was that E₃ dissociates from unheated 8S RE₃ and heat-transformed 5S RE₃ at a much faster rate than E₂ from RE₂;In the mature ovariectomized rat a slow dissociating 5S receptor estrogen complex is found in nuclei 1 h after injection of [³H]E₂ or [³H]E₃. In vitro dissociation of these 2 estrogens from this nuclear bound receptor formed in vivo takes place at rates similar to those from heat-transformed cytosolic RE₂ or RE₃ complexes.Addition of pyridoxal 5'-phosphate (PLP) to the slow-dissociating heat-transformed 5S estrogen receptor complexes causes rapid dissociation of E₂ or E₃; this effect is dose-dependent and is not due to disruption of 5S dimers, since after PLP addition RE₂; and RE₃ sediment unchanged as 5S dimers.The presence of a large excess of non-radioactive 4S RE₃ does not interfere with the temperature induced rapid transition of 4S R[³H]E₂ complexes from the state with fast into a state with slow E₂ dissociation kinetics.A model is presented to explain the temperature induced biphasic estrogen dissociation from the receptor. It is proposed that the low affinity 4S RE₂ monomer undergoes a temperature and estrogen dependent conformation change, such that the ligand is “locked” into the receptor's binding site. This conformational change results in the formation of a high affinity 4S monomer from which estrogen dissociates at a slower rate. This reaction is independent from subsequent 4S to 5S dimerization (transformation). The different rates of ligand dissociation from the low and high affinity 4S receptors reflect the different interactions (hydrophobic and hydrogen bonding) of E₂ and E₃ with the estrogen binding domain.     

Bacillus megaterium, KM beta-galactosidase: purification by affinity chromatography and characterization of the active species

The enzyme β-galactosidase from Bacillus megaterium, strain KM has been purified by affinity chromatography. The enzyme was found to have a dimeric subunit structure, with the monomer having a molecular weight of 120,000. The K_eq of the monomer-dimer equilibrium was strongly shifted towards dissociation in the isolated state. Inclusion of 5% sucrose in the buffer (and maintenance of the temperature at 5 °) minimized this dissociation. Molecularly homogeneous monomer and dimer could be prepared on sucrose gradients. The dimer was determined to have an S_20,w of 8, while the monomer had an S_20,w of 3. The amino acid composition was found to be similar to that of the E. coli β-galactosidase although significant differences occur. The activity of the monomer was studied by both urea-denaturation experiments and by immobilization of the monomer on Sepharose-4B. The monomer, bound to Sepharose-4B, was found to be inactive but still capable of binding the inhibitor thio-methyl galactoside. Activity was reconstituted by adding free monomer, in 8 M urea, to the Sepharose-bound monomer, followed by removal of the urea by dialysis. In addition, free monomers from E. coli β-galactosidase were found to form active hybrids with Sepharose-bound B. megaterium β-galactosidase monomers. We conclude on the basis of these studies that the free monomer is inactive, and that the dimer is the active species, in marked contrast to E. coli β-galactosidase where only the tetrameric form is active.