Phaseotoxin A: an antimetabolite from Pseudomonas phaseolicola.

Phaseotoxin, the exotoxin of the bean pathogen $\text{Pseudomonas phaseolicola}$, has been resolved into four biologically active fractions by DEAE cellulose chromatography. Phaseotoxin A, the toxin in the first fraction decomposed to give glutamic acid and inorganic orthophosphate only, and was chromatographically identical with synthetic N-phosphoglutamic acid. Like phaseotoxin A, the synthetic compound inhibits ornithine carbamoyltransferase and induces chlorosis in bean leaves. To our knowledge this is the first report of occurrence of an N-phosphorylated primary amine in nature.     

Unmasking of an essential thiol during function of the membrane-bound enzyme II of the phosphoenolpyruvate β-glucoside phosphotransferase system of Escherichia coli

β-Glucoside transport by phosphoenolpyruvate-hexose phosphotransferase system in Escherichia coli is inactivated in vivo by thiol reagents. This inactivation is strongly enhanced by the presence of transported substrates. In a system reconstituted from soluble and membrane-bound components, only the particulate component, the membrane-bound enzyme II^bgl appeared as the target of N-ethylmaleimide inactivation. The same feature was found in the case of methyl-α-d-glucoside uptake via enzyme II^glc.It is shown that the sensitizing effect of substrates is specific and not generalized, methyl-α-d-glucoside only sensitizes enzyme II^bglc and $\text{p-}\text{nitrophenyl-β-}\text{d}\text{-glucoside}$ only sensitizes enzyme II^bgl towards N-ethylmaleimide inactivation.The inactivation of enzyme II^bgl by thiol reagents is also promoted in vivo by fluoride inhibition of phosphoenolpyruvate synthesis. In toluene-treated bacteria, the presence of phosphoenolpyruvate protects against inactivation by thiol reagents of $\text{p-}\text{nitrophenyl-β-}\text{d}\text{-glucoside}$ phosphorylation. Both results suggest that the inactivator resistent form of enzyme II^bgl is an energized form of the enzyme.     

On the defect of a dCMP hydroxymethylase mutant of bacteriophage T4 showing enzyme activity in extracts

Infection by $\text{L}\text{13}$, a temperature-sensitive mutant of gene 42 of phage T4, the structural gene for dCMP hydroxymethylase, previously was shown not to form T4 DNA at nonpermissive temperatures. Yet the enzyme activity was found in extracts. Since inactivation of the enzyme was not reversible, we have examined acid-soluble extracts of cells infected at nonpermissive temperature by $\text{tsL}\text{13}$ for 5-hydroxymethyldCMP in order to determine whether the enzyme functioned $\text{in vivo}$. A double mutant of $\text{tsL13}$ and $\text{amB}\text{24}$ (5-hydroxymethyldCMP kinase) did not form the nucleotide at nonpermissive temperature, but the control, $\text{amB}\text{24}$, formed large quantities. From these results and previous temperature-shift studies it is suggested that the enzyme is normally activated to function $\text{in vivo}$ between 5 and 8 minutes after infection.     

Structural changes in (Na+ + K+)-ATPase accompanying detergent inactivation

Structural changes in the purified $\text{(}\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ accompanying detergent inactivation were investigated by monitoring changes in light scattering, intrinsic protein fluorescence, and tryptophan to β-parinaric acid fluorescence resonance energy transfer. Two phases of inactivation were observed using the non-ionic detergents, digitonin, Lubrol WX and Triton X-100. The rapid phase involves detergent monomer insertion but little change in protein structure or little displacement of closely associated lipids as judged by intrinsic protein fluorescence and fluorescence resonance energy transfer. Lubrol WX and Triton X-100 also caused membrane fragmentation during the rapid phase. The slower phase of inactivation results in a completely inactive enzyme in a particle of 400 000 daltons with 20 mol/mol of associated phospholipid. Fluorescence changes during the course of the slow phase indicate some dissociation of protein-associated lipids and an accompanying protein conformational change. It is concluded that non-parallel inhibition of $\text{(}\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ and $\text{p-}\text{nitrophenylphosphate}$ activity by digitonin (which occurs during the rapid phase of inactivation) is unlikely to require a change in the oligomeric state of the enzyme. It is also concluded that at least 20 mol/mol of tightly associated lipid are necessary for either $\text{(}\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ or $\text{p-}\text{nitrophenylphosphatase}$ activity and that the rate-limiting step in the slow inactivation phase involves dissociation of an essential lipid.     

Nitrate reductase from Azotobacter chroococcum. Inactivation by oxidizing agents and reactivation with dithioerythritol

Treatment of a partially purified nitrate reductase preparation from the aerobic bacterium $\text{Azotobacter chroococcum}$ with a variety of oxidizing agents, such as glutathione, ferricyanide and illuminated flavins, results in inactivation of the enzyme. Independently of the mode of inactivation, incubation in the presence of dithioerythritol causes almost full recovery of nitrate reductase activity. Our data suggest that $\text{Azotobacter}$ nitrate reductase might be regulated through an interconversion process between an oxidized inactive form and a reduced active one.     

Oxygen poisoning of NAD biosynthesis: a proposed site of cellular oxygen toxicity.

Quinolinate phosphoribosyl transferase was rapidly inactivated in $\text{Escherichia}\text{coli}$ exposed to hyperbaric oxygen. The enzyme is essential for de novo biosynthesis of NAD in $\text{E.}\text{coli}$ and man. Because of its sensitivity and essentiality, inactivation of this enzyme is proposed as a significant mechanism of cellular oxygen toxicity. Niacin which enters the NAD biosynthetic pathway below the oxygen-poisoned enzyme provided significant protection against the decrease in pyridine nucleotides and the growth inhibition from hyperoxia in $\text{E.}\text{coli}$ and could be useful in cases of human oxygen poisoning.     

4-ethenylidene steroids as mechanism-based inactivators of 3β-hydroxysteroid dehydrogenases

The synthesis of 4-ethenylidene-5α-androstane-3β, 17β-diol ($\text{5}$) and of 4-ethenylidene-5α-androstane-3,17-dione ($\text{4}$) is described. Compound $\text{5}$ is a competitive inhibitor of solubilized bovine microsomal adrenal Δ⁵-3β-hydroxysteroid dehydrogenase, with Ki =2.7μM, and is converted by the enzyme to the corresponding 3-ketone. Compound $\text{4}$ shown to irreversibly inactivate the enzyme in a time-dependent manner ($\text{t}\text{1}\text{2}\text{=31}\text{min}\text{; 55μ}\text{M}\text{;}\text{pH}\text{=7.0}$). The substrate, dehydroepiandrosterone, protects against inactivation by compound $\text{4}$. In contrast, compound $\text{5}$ is not oxidized at the 3-position by the 3β-(and 17β)-hydroxysteroid dehydrogenase from $\text{P. testosteroni}$, but is oxidized at the 17-position. Nevertheless, the 4-ethenylidene-3,17-diketone ($\text{4}$) causes irreversible time-dependent inactivation ($\text{t}\text{1}\text{2}\text{=28}\text{min}\text{; 64μ}\text{M}\text{;}\text{pH}\text{=7.0}$) when incubated directly with this bacterial enzyme, acting as an affinity label.     

The thermal lability of adenylate cyclase: Mechanisms of stabilization

The thermal inactivation of adenylate cyclase was investigated in human lymphocytes and in the N-protein deficient cyc-S49 mouse lymphoma cell line. The enzyme is rapidly inactivated at 37C with a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 5.5 and 4.5 min respectively in human and cyc^? membranes. Thermal inactivation is prevented by at least two mechanisms. The first mechanism involves ATP which stabilizes adenylate cyclase in a concentration dependent manner similar to the K_m of ATP for cAMP formation. However, the inhibition of inactivation does not require Mg⁺⁺ while the enzyme catalysis of ATP to cAMP does. The second mechanism involves substances which activate the enzyme. The human lymphocyte enzyme is equally stabilized by either NaF, GppNHp, or forskolkin. In contrast, the cyc^? enzyme is fully stabilized by forskolin but only partially stabilized by NaF. When human erythrocyte N-protein extract is added to cyc^? membranes, NaF fully stabilizes the enzyme. These data suggest that an activated N-protein is instrumental in stabilizing adenylate cyclase and that there is some N-protein component in cyc^? membranes through which NaF may be exerting its stabilizing action.     

Mn++-specific reactivation of EDTA inactivated alpha-isopropylmalate synthase from Alcaligenes eutrophus H 16.

The α-isopropylmalate synthase (EC 4.1.3.12) from $\text{Alcaligenes}\text{eutrophus}\text{H 16}$ was inactivated by EDTA in a time-dependent reaction. Only the addition of Mn⁺⁺ plus dithiothreitol could restore the activity. The substrate, α-ketoisovalerate, prevented the inactivation; the feedback inhibitor, leucine, and it's antagonist, valine, increased the rate of inactivation. Except for α,α′-bipyridyl, chelating reagents, other than EDTA had no effect on the enzyme stability. It is suggested that the α-isopropylmalate synthase is a metallo enzyme - the evidence points to Mn⁺⁺ as the metal ion - and that this enzyme uses a mechanism of catalysis which differs from that of the analogous malate synthase (EC 4.1.3.2) and citrate synthase (EC 4.1.3.4).     

A mutation affecting the valine sensitivity of the acetohydroxyacid synthase III isoenzyme in E. coli K-12

The $\text{ilv-751}$ mutation (obtained by $\text{mu}$ phage mediated mutagenesis) affects the sensitivity to valine inhibition of the acetohydroxy acid synthase III isoenzyme of $\text{E. coli}$ K-12, as shown by constructing multiple mutants containing the $\text{ilv-751}$ mutation and only one of the genes for the expression of the three acetohydroxy acid synthase isoenzymes, at once. The mutation is dominant. This suggests that the phenotype of $\text{ilv-751}$ mutation is not caused by inactivation of a gene concerned with the expression of the AHASIII enzyme, consequent to prophage insertion into that locus.     

Inactivation of ribulosebisphosphate carboxylase/oxygenase from rhodospirillum rubrum and spinach with the new affinity label 2-bromo-1,5-dihydroxy-3-pentanone 1,5-bisphosphate

In an attempt to identify the active-site base believed to initiate catalysis by ribulosebisphosphate carboxylase, we have synthesized 2-bromo-1, 5-dihydroxy-3-pentanone 1,5-bisphosphate, a reactive analogue of a postulated intermediate of carboxylation. Although highly unstable, this compound can be shown to inactivate the carboxylases from both $\text{Rhodospirillum}\text{,}\text{rubrum}$ and spinach rapidly and irreversibly. Inactivation follows pseudo first-order kinetics, shows rate saturation and is greatly reduced by saturating amounts of the competitive inhibitor, 2-carboxyribitol 1,5-bisphosphate. The incorporation of reagent, quantified by reducing the modified carboxylases with [³H]NaBH₄, shows that inactivation results from the modification of approximately one residue per catalytic subunit of the $\text{Rhodospirillum}\text{,}\text{rubrum}$ enzyme and less than one residue per protomeric unit of the spinach enzyme.     

Essential arginyl residues in thymidylate synthetase.

Thymidylate synthetase from amethopterin-resistant $\text{Lactobacillus}$$\text{casei}$ is rapidly and completely inactivated by 2,3-butanedione in borate buffer, a reagent that is highly selective for the modification of arginyl residues. The reversible inactivation follows pseudo-first order kinetics and is enhanced by borate buffer. dUMP and dTMP afford significant protection against inactivation while (±)-5,10-methylenetetrahydrofolate and 7,8-dihydrofolate provide little protection. Unlike native enzyme, butanedione-modified thymidylate synthetase is incapable of interacting with 5-fluoro-2′-deoxyuridylate and 5,10-(+)-methylenetetrahydrofolate to form stable ternary complex. The results suggest that arginyl residues participate in the functional binding of dUMP.     

Proteolytic inactivation of a pentafunctional enzyme conjugate: coordinate protection by the first substrate.

The $\text{arom}$ pentafunctional enzyme conjugate of $\text{Neurospora crassa}$ was exposed to trypsin, chymotrypsin, or a protease preparation from $\text{Neurospora}$ in the presence and absence of the first substrate, 3-deoxy-D-$\text{arabino}$-heptulosonate 7-phosphate. It was found that the first substrate coordinately protects all five activities from proteolytic inactivation, which indicates a conformational change induced by this compound. In addition, the data presented are consistent with the “domain” theory of conjugate structure. It is also argued that coordinate protection may be of physiological significance.     

Changes in accessibility of the membrane bound transport enzyme glucose phosphotransferase of E. coli to protein group reagents in presence of substrate or absence of energy source

The inactivation of α-methyl-D-glucoside transport in $\text{E. coli}$ by N-ethyl-maleimide and 1-fluoro-2,4-dinitrobenzene is strongly enhanced by the presence of substrate or of an inhibitor of phosphoenol pyruvate synthesis. It is demonstrated in the case of N-ethylmaleimide that the target of the inhibition is the membrane bound component of the phosphoenol pyruvate glucose phospho-transferase system: enzyme II. Enzyme II could exist, in course of the transport, in two alternate conformational states: an energized and a deenergized state, the energized one being protected against inactivation by N-ethylmaleimide.     

Inhibition of β-lactamase-I by 6-β-sulfonamidopenicillanic acid sulfones: Evidence for conformational change accompanying the inhibition process

A number of 6-β-sulfonamidopenicillanic acid sulfones were examined for their ability to inhibit Bacillus cereus$\text{569}\text{H}$ β-lactamase I. Among these, 6-β-trifluoromethane sulfonamidopenicillanic acid sulfone was found to be the most potent inhibitor, effecting rapid and irreversible inactivation of the enzyme. Optical rotatory dispersion and differential scanning calorimetry were employed to probe the possible conformational changes accompanying the inactivation of B. cereus$\text{569}\text{H}$ β-lactamase 1 by 6-β-trifluoromethane sulfonamidopenicillanic acid sulfone. Optical rotatory dispersion measurements indicated the presence of approximately 29 and 17% helical structure in the native and inactivated enzyme, respectively. Differential scanning calorimetry determinations revealed that the inactivated enzyme was less thermostable than the native β-lactamase. The temperatures of maximum heat absorption were 48.4(±0.5) and 57.4(±0.1)°C for the inactivated and the native enzyme, respectively. Extensive conformational changes accompanying the interaction of the enzyme with the inhibitor may be responsible for the irreversible loss in the catalytic activity.     

Cloning and expression in Streptomyces lividans of a paromomycin phosphotransferase from Streptomyces rimosusforma paromomycinus

The paromomycin producing organism $\text{Streptomyces}$$\text{rimosus}$$\text{forma paromomycinus}$ is resistant to this antibiotic and contains a phosphotransferase which inactivates paromomycin. The gene encoding this enzyme has been inserted in the $\text{Streptomyces}$ vector pIJ702 and then cloned in $\text{Streptomyces}$$\text{lividans}$, selecting for paromomycin-resistance. Three plasmids have been isolated and one of them, pMJ1, contains a 2.2 kb insert with a single HindIII restriction site. Insertion of foreign DNA in this site blocks the expression of the phosphotransferase enzyme indicating that it is within the cloned gene. These findings provide a new dominant selective marker for $\text{Streptomyces}$ cloning vectors with the versatility of insertional inactivation.     

Purification of the insecticidal toxin from the parasporal crystal of Bacillus thuringiensis subsp. kurstaki

The insecticidal toxin of $\text{Bacillus}\text{thuringiensis}$ subsp. $\text{kurstaki}$ was isolated from parasporal crystals. The toxin, which is stable for several months, is a glycoprotein with an apparent molecular weight of 68,000 that is generated upon solubilization and activation of a higher molecular weight protoxin (MW_app = 1.3 × 10⁵) at alkaline pH. The toxin was purified by gel filtation and anion exchange chromatography and its molecular weight was established by gel filtration chromatography and SDS polyacrylamide gel electrophoresis.     

Evidence for an essential lysine residue on the phosphoribosyl transferase subunit of the anthranilate synthetase-anthranilate 5-phosphoribosyl-pyrophosphate phosphoribosyltransferase enzyme complex from Salmonella typhimurium.

Pyridoxal 5′-phosphate reacts with the anthranilate synthetase-phosphoribosyltransferase enzyme complex of $\text{Salmonella typhimurium}$ to inhibit PR transferase activity. Glutamine-dependent anthranilate synthetase is not affected. Spectral and kinetic data suggest that the inactivation results from the modification of an essential lysine residue which interacts with 5-phosphoribosyl 1-pyrophosphate.     

Ornithine decarboxylase protein diversity and activity modulation in HTC cells

Ornithine decarboxylase of HTC cells was chromatographically separated into three ionically distinct but kinetically similar forms of this protein. The sequential appearance of these ornithine decarboxylase species during enzyme induction, and the accumulation of normally minor species under conditions that stabilize this enzyme, suggest that these represent modifications that are associated with the extremely rapid turnover of this protein $\text{in vivo}$. These forms may also be differentially active or unequally distributed $\text{in vivo}$ as indicated by the selective inactivation of one of the forms by short exposure to α-difluoromethylornithine.     

Irreversible inactivation of rat liver tyrosine aminotransferase

Homogenates prepared from rat livers irreversibly inactivate tyrosine aminotransferase, both endogenous and purified exogenous enzyme, in the presence of certain compounds which bind to pyridoxal 5′-P. The rate of inactivation ranged from a half-life of 0.72 to greater than 15 hr. The pyridoxal 5′-P binding compounds may be considered to be structural analogs for α-ketoglutarate or l-tyrosine, both of which are substrates for the enzyme. l-Cysteine and l-DOPA are the most effective compounds tested of each of the two structural analog classes, respectively. Absence of the carboxyl group from l-cysteine or l-DOPA has little effect on the half-life of the enzyme, whereas absence or substitution of the amino group results in an increased enzyme half-life. Absence of the —SH group from l-cysteine or of the 3′-OH group from l-DOPA results in little or no inactivation of the enzyme ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ increased to greater than 15 hr). Semicarbazide and hydroxylamine have little effect on the stability of the enzyme. Addition of pyridoxal 5′-P to homogenates incubated with l-cysteine or l-DOPA inhibits the inactivation of the enzyme. However, the addition of cofactor to inactivated enzyme does not restore lost activity.There is a disappearance of antigenic cross-reacting material during inactivation of the enzyme. This loss of specific cross-reacting material occurs at a slower rate than the loss of enzyme activity, indicating that enzymatic activity is lost prior to loss of antigenic recognition. A three-step proposal is presented to explain the data observed in which the first step is a reversible loss of pyridoxal 5′-P from the enzyme, followed by a specific irreversible inactivation of the enzyme, and ending with nonspecific proteolysis or degradation of the inactivated enzyme molecules.