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At least 19 recordsLinked to original sources

PubMed1975

Aminoglycoside 3'-phosphotransferase III, a new phosphotransferase. Resistance mechanism.

The aminoglycoside phosphotransferase of Pseudomonas aeruginosa 21-75 was purified by affinity chromatography using dibekacin-Sephadex 4B or lividomycin A-Sepharose 4B followed by DEAE Sephadex A-50 chromatography. It had activities of both the known aminoglycoside 3'-phosphotransferases I and II, and transferred phosphate from ATP to the 3'-hydroxyl group of kanamycin A, ribostamycin and butirosin A and 5-hydroxyl group of lividomycin A. This enzyme was designated aminoglycoside 3'-phosphotransferase III. It showed strong substrate inhibition by kanamycin A and ribostamycin when their concentration exceeded 6 muM. Purification and characterization of this enzyme are reported.

Aminoglycosides↗

PubMed1977

Genetic evidence for the common identity of glucose-6-phosphatase, pyrophosphate-glucose phosphotransferase, carbamyl phosphate-glucose phosphotransferase and inorganic pyrophosphatase.

We demonstrate that glucose-6-phosphatase, pyrophosphate-glucose phosphotransferase, carbamyl phosphate-glucose phosphotransferase and inorganic pyrophosphatase activities are deficient in livers of patients with type I glycogen storage disease. This provides strong genetic evidence that these enzymatic activities reside in a single protein or share a common polypeptide chain.

Carbamyl Phosphate↗

PubMed1977

Effects of deoxycholate and phospholipase A2 on choline and ethanolamine phosphotransferases of chicken brain microsomes.

Ethanolamine phosphotransferase (EC 2.7.8.1) and choline phosphotransferase (EC 2.7.8.2) activities were assayed in fresh microsomes from adult chicken brains with either diacylglycerols or alkylacylglycerols. Pretreatment of microsomes with 1.25 mM sodium deoxycholate, a concentration less than the critical micelle concentration, produced a slight inhibition of choline phosphotransferase activity. A deoxycholate concentration (5.0 mM) greater than the critical micelle concentration (3.0 mM) decreased the choline phosphotransferase activity by more than 70% but had no effect on ethanolamine phosphotransferase activity. Inclusion of 1.25 mM deoxycholate in the assay medium decreased choline phosphotransferase activity 35% but increased ethanolamine phosphotransferase activity 50%. The deoxycholate appeared to inactive the choline phosphotransferase. Phospholipase A2 (Vipera russelli) treatments of microsomes removed phosphoglycerides and decreased both phosphotransferase activities to a similar extent. Decreased activities are probably due to disruption of the membrane structure. Choline and ethanolamine phosphotransferase activities are apparently in different enzymes which lack specificity for the type of diglyceride. Thus, the systematic names should include 1,2-diradyl-sn-glycerol instead of 1,2-diacyl-sn-glycerol.

Animals↗

PubMed1976

Thymidine phosphotransferase and nucleotide phosphohydrolase of the fern Asplenium nidus. General properties and inhibition by adenosine 3':5'-cyclic monophosphate.

1. Extracts of several plant species contained nucleoside-AMP phosphotransferase activity. The ratio of activity with thymidine to that with uridine as nucleoside substrate was essentially constant, both between species and throughout plant development. Evidence is presented that the total thymidine-AMP phosphotransferase activity of the leaves of Asplenium nidus (bird's-nest fern) and of Helianthus tuberosus (Jerusalem artichoke) increases during maturation. 2. Thymidine-AMP phosphotransferase was purified 22-fold from a very rich source of this activity, extracts of A. nidus. 3. A broad specificity towards both nucleoside and nucleoside 5'-monophosphate substrates is displayed by this preparation, and the evidence suggests that all could be due to a single enzyme. 4. Nucleosides that act as substrates will also inhibit phosphotransfer to other nucleosides, with Ki values close to the corresponding Km values found when utilized as substrates. 5. Ca2+-activated ATP phosphohydrolase was separated from the phosphotransferase by differential complexing to Blue Dextran in the presence of urea, whereas an AMP phosphohydrolase activity was closely associated with thymidine-AMP phosphotransferase through all separation techniques used. 6. Metal ions did not activate either of the latter two activities, and 1,10-phenanthroline was found to inhibit the phosphotransferase. 7. Km values for AMP for the respective activities were 0.11 mM (thymidine phosphotransferase) and 0.20 mM (AMP phosphohydrolase) and for thymidine (phosphotransferase only) 0.88 mM. 8. 3':5'-Cyclic AMP was found to inhibit both phosphotransferase and AMP phosphohydrolase activities, with Ki values of 0.056 mM and 0.15 mM respectively. It is suggested that this inhibitor would be of value in revealing the existence of thymidine kinase in plant extracts with high thymidine phosphotransferase activity.

Adenosine Monophosphate↗

PubMed1979

Amikacin resistance associated with a plasmid-borne aminoglycoside phosphotransferase in Escherichia coli.

Enzymatic phosphorylation of amikacin has not been reported previously in gram-negative bacteria. We found that extracts of MP1, a mutant of Escherichia coli JR66/W677 that is resistant to amikacin, were able to phosphorylate this aminoglycoside more rapidly than were extracts of the parental strain. Conjugal transfer of resistance from MP1 to a recipient strain was accompanied by acquisition in the transconjugants of amikacin phosphotransferase activity and of a 57-megadalton plasmid present in the donor. Partial purification of the phosphotransferase activity on amikacin-Sepharose 4B yielded an enzyme with a substrate spectrum similar to that of the 3'-neomycin-kanamycin phosphotransferase II found E. coli, except that it was also active against amikacin. A mutant of MP1, MP5, had increased susceptibility to amikacin and reduced phosphotransferase activity. MP9, a mutant MP5, was more resistant to amikacin and had increased phosphotransferase activity. The mutations leading to these alterations of amikacin susceptibility and amikacin phosphotransferase activity were transferable with the same plasmid that was associated with amikacin resistance and phosphotransferase activity in MP1. These studies demonstrate that resistance to amikacin in a laboratory strain of E. coli is due to an aminoglycoside phosphotransferase coded by a transferable plasmid-borne gene.

Amikacin↗

PubMed1975

Viral stimulation of choline phosphotransferase in spleen microsomes.

Choline phosphotransferase and phosphatidyl ethanolamine methyltransferase enzymatic activities (nmoles phosphatidyl choline/min/mg protein) have been determined in spleen microsomes of Rauscher virus infected balb/c male mice at 5, 10, 14, and 21 days following inoculation of the virus. There is a significant stimulation of the choline phosphotransferase activity in the virus infected spleens with the peak of activity at about 10 days of viral infection. The specific activity of choline phosphotransferase is 10 times that of the phosphatidyl ethanolamine methyltransferase at 10 days of viral infection. There is a 51-fold increase over controls for the total microsomal choline phosphotransferase at 14 days of viral infection and only an 18-fold increase over controls for the phosphatidyl ethanolamine methyltransferase activity. There is a significant (P less than 0.001) increase over controls in the concentration of total phospholipid-P, phosphatidyl choline-P, and phosphatidyl choline-P fractions as separated by argentation chromatography of microsomes from spleens of mice infected with Friend virus of Rauscher virus for 14 days. The choline phosphotransferase and phosphatidyl ethanolamine methyltransferase specific activities in liver microsomes of 14 day Friend and/or Rauscher virus are unaltered during viral infection.

Animals↗

PubMed1976

Enzymes of phospholipid synthesis in Bacillus Calmette-Guerin induced rabbit alveolar macrophage. Characterization and localization of cytidine diphosphocholine phosphotransferase and monoacylphospholipid acyltransferase.

The rabbit alveolar macrophage is capable of renewing its plasma membrane by at least two metabolic pathways. It contains (1) a monoacylphospholipid acyltransferase, which catalyzes the synthesis of diacylphospholipids by recycling monoacylphospholipids produced by the action of phospholipases and (2) a cytidine diphosphocholine phosphotransferase (CDPcholine phosphotransferase), which catalyzes the last step in the synthesis de novo of diacylglycerophosphocholine. These activities have been characterized in the cell homogenate with respect to time, protein, pH optimum (for CDPcholine phosphotransferase), substrate specificity (for monoacylphospholipid acyltransferase) and cation requirement ( for CDPcholine phosphotransferase). Monoacylphospholipid acyltransferase activity is localized solely in the endoplasmic reticulum. On the other hand, the CDPcholine phosphotransferase activity can be measured in the endoplasmic reticulum and in the plasma membrane, characterized by both differential and gradient sedimentation techniques. In addition to the normal route of phospholipid synthesis in the endoplasmic reticulum, the rabbit alveolar macrophage may thus possess the capacity for in situ synthesis of phospholipids of plasma membrane as a mechanism for membrane renewal following phagocytosis.

Acyltransferases↗

PubMed1979

Stimulation by polyamines of carbamylphosphate:glucose phosphotransferase and glucose-6-phosphate phosphohydrolase activities of multifunctional glucose-6-phosphatase.

The effects of added polyamines on carbamylphosphate (carbamyl-P):glucose phosphotransferase and glucose-6-phosphate (Glc-6-P) phosphohydrolase activities of rat hepatic D-Glc-6-P phosphohydrolase (EC 3.1.3.9) of intact and detergent-treated microsomes have been investigated. With the former preparation, in the presence of 1.4 mM phosphate substrate and 90 mM D-glucose (phosphotransferase), 1 mM spermine, spermidine, and putrescine activated Glc-6-P phosphohydrolase 67%, 57%, and 35%, respectively. Carbamyl-P:glucose phosphotransferase, under comparable conditions, was activated 57%, 34%, and 18%. NH+4 (0.25--5.0 mM) produced at best but a minor activation (0--14%), while poly(L-lysine) (Mr = 3400; degree of polymerization 16) equimolar relative to other polyamines with respect to ionized free amino groups activated the hydrolase 358% and the transferase 222%. Treatment of microsomes with the detergent deoxycholate reduced, but did not abolish, polyamine-induced activation. The stimulatory effects of polyamines persisted in the presence of excess catalase, indicating their independence from H2O2 formation; and were eliminated in the presence of Ca2+. Kinetic analysis revealed that all tested polyamines decreased the apparent Michaelis constant values for carbamyl-P and Glc-6-P, but had no effect on the Km for glucose. Poly(L-lysine) increased the V value for both Glc-6-P phosphohydrolase and apparent V values for phosphotransferase extrapolated to infinite concentrations of either carbamyl-P or glucose. The other tested polyamines elevated only this last velocity parameter. It is proposed that a major mechanism by which polyamines activate glucose-6-phosphatase-phosphotransferase is through their electrostatic interactions with phospholipids of the membrane of the endoplasmic reticulum of which this enzyme is a part. Conformational alterations thus induced may in turn affect catalytic behavior. It is suggested that polyamines, or similar positively charged peptides, might participate in the cellular regulation of synthetic and hydrolytic activities of glucose-6-phosphatase.

Ammonium Chloride↗

PubMed1979

Levels of true thymidine kinase and nucleoside phosphotransferase in two strains of Tetrahymena pyriformis under different growth conditions.

Activities of typical thymidine kinase and nucleoside phosphotransferase are both present in logarithmically growing tetrahymena pyriformis, GL-1 and ST strains, contrary to previous reports. 2. Activities of thymidine kinase and nucleoside phosphotransferase are also found in both GL-1 and ST strains grown in the defined medium, PPL medium and Neff's medium. 3. The specific activities of both enzymes are very much influenced by the growth state. Both the specific activities of thymidine kinase and nucleoside phosphotransferase decrease steadily from the start of the experiments when the cell numbers were about 2-3 x 10(4) cells/ml in the PPL medium, while in the Neff's medium, the specific activities of thymidine kinase increase up to when the cell numbers reached 3-5 x 10(5) cells/ml and then decreased, but the specific activities of nucleoside phosphotransferase continuously decreased when the cell concentrations were 2-6 x 10(4) cells/ml. 4. In the PPL medium, the final cell numbers reached are about 6.5 x 10(5) cells/ml, while in the Neff's medium, the cell numbers increase further (to about 2 x 10(6) cells/ml). 5. No striking difference in activities of thymidine kinase and nucleoside phosphotransferase was observed when the cells were transferred from the defined medium to the Neff's medium, contrary to that reported by others for the activity of thymidylate synthetase.

Animals↗

PubMed1979

Induction of the phosphoenolpyruvate: hexose phosphotransferase system associated with relative anaerobiosis in an obligate aerobe.

Arthrobacter pyridinolis possesses alternative transport systems for D-fructose: a respiration-coupled transport system whereby D-fructose transport occurs with concomitant oxidation of L-malate, and a phosphoenolpyruvate: D-fructose phosphotransferase system. Studies of D-fructose uptake by whole cells in the presence and absence of cyanide demonstrate that respiration-coupled transport is used almost exclusively during the first half of logarithmic growth, after which it accounts for only 15-20% of D-fructose uptake. Phosphotransferase levels are low during log phase, peak during late log, and then slowly decline. In a mutant of A. pyridinolis which requires delta-aminolevulinic acid for growth, the growth rate, cell cytochrome content, and activity of the respiration-coupled transport system increased with increasing concentrations of delta-aminolevulinic acid up to 50 microgram/ml. By contrast, phosphotransferase activity was highest in cells grown on limiting delta-aminolevulinic acid. L-Malate, which stimulates respiration-coupled transport, repressed the phosphotransferase system. The respiratory activity and the ability to release CO2 from internalized d-fructose was consistently low in D-fructose-grown cells. A cyanide-resistant cytochrome, tentatively identified as cytochrome d, appeared in the late exponential phase of growth. Isocitrate lyase activity, required for aerobic growth of this organism, declined markedly during the late exponential phase. Thus the phosphotransferase system is maximally induced, in this obligate aerobe, under conditions of relative anaerobiosis during which metabolism is primarily fermentative.

Aerobiosis↗

PubMed1992

Utilisation of glycerol and glycerol 3-phosphate is differently affected by the phosphotransferase system in Bacillus subtilis.

Glycerol and glycerol 3-phosphate uptake in Bacillus subtilis does not involve the phosphotransferase system. In spite of this, B. subtilis mutants defective in the general components of the phosphotransferase system, EnzymeI or Hpr, are unable to grow with glycerol as sole carbon and energy source. Here we show that a Hpr mutant can grow on glycerol 3-phosphate and that glycerol 3-phosphate, but not glycerol, can induce glpD encoding glycerol-3-phosphate dehydrogenase. Induction of glpD also requires the glpP gene product which is a regulator of all known glp genes. Thus the phosphotransferase system general components do not interfere with the overall regulation of the glp regulon. Revertants of a Hpr mutant which can grown on glycerol carry mutations closely linked to the glp region at 75 degrees on the B. subtilis chromosomal map. This region contains the glpP, the glpFK and the glpD operons. The glpFK operon encodes the glycerol uptake facilitator (glpF) and glycerol kinase (glpK). The present results demonstrate that one of these genes, or their gene products, is the target for phosphotransferase system control of glycerol utilisation. Furthermore we conclude that utilisation of glycerol and glycerol 3-phosphate is differently affected by the phosphotransferase system in B. subtilis.

Bacillus subtilis↗

PubMed1977

Initial characterization of hexose and hexitol phosphoenolpyruvate-dependent phosphotransferases of Staphylococcus aureus.

The phosphoenolpyruvate sugar phosphotransferases of Staphylococcus aureus were surveyed biochemically to determine substrate range, inducibility and constitutivity, and requirements for soluble sugar-specific proteins. The substrate range is similar to that of the phosphotransferases of enteric bacteria, but the staphylococcal mannose and sorbitol systems are very inefficient. In addition, S. qureus has phosphotransferase activities for lactose and sucrose. The systems tested fell into two broad classes. Sugars for which there was substantial constitutive activity (fructose, mannose, sucrose, and glucose and its nonmetabolized analogues) did not require sugar-specific soluble factors for phosphorylation. Only in the case of fructose did growth in the presence of these constitutive sugars induce the corresponding phosphotransferase activity to higher levels. Kinetic experiments with each of these constitutive sugars yielded biphasic Hofstee plots; i.e., the kinetics were not characteristic of single enzymes. Preliminary experiments suggest that sucrose phosphorylation may involve the glucose and/or fructose systems. Truly inducible sugar phosphotransferase systems represent a second class; those for lactose and mannitol are the only members thus far identified. These systems are absent from uninduced cells, require soluble sugar-specific factors, and exhibit linear Hofstee plots. Sorbitol is apparently transported very poorly by intact cells but is an inducer of the mannitol system; it is phosphorylated efficiently in vitro by extracts of cells grown on either hexitol, but is taken up by intact cells at 0.1% of the mannitol rate.

Cell Membrane↗

PubMed1977

Xylitol and D-arabitol toxicities due to derepressed fructose, galactitol, and sorbitol phosphotransferases of Escherichia coli.

d-Arabitol was observed to be toxic to many laboratory strains of Escherichia coli K-12, and xylitol was found to be toxic to an existing E. coli C mutant strain. Fructose-specific components of the phosphoenolpyruvate:sugar phosphotransferase system are required for xylitol toxicity. Selection for xylitol resistance results in Fru(-) strains blocked in fructose phosphotransferase. Introduction of the ptsF or ptsI mutation into a xylitol-sensitive strain eliminates sensitivity. [(14)C]fructose uptake experiments imply that the mutation to xylitol sensitivity, which is co-transducible with ara and leu, results in derepression of normally inducible fructose phosphotransferase. Wild-type strains also become xylitol sensitive if induced by (and then removed from) fructose. Xylitol toxicity is prevented by fructose in both wild-type and mutant strains. Circumstances causing xylitol, a new food additive, to become toxic to an otherwise insensitive wild-type organism have not been reported previously. The d-arabitol-sensitive laboratory strains are galactitol (dulcitol) utilizers, although most other strains are not. Selection for d-arabitol resistance results in Gat(-) strains blocked in a constitutive galactitol-specific component of the phosphotransferase system. A mutation causing d-arabitol sensitivity occurred many years ago in AB284, the parent of AB311, AB312, AB313, and many other strains. d-Arabitol sensitivity also occurs in sorbitol-constitutive strains and is shown, like the previous two instances of pentitol toxicities, to result from a constitutive phosphotransferase, which is blocked in mutants selected for resistance.

Arabinose↗

PubMed1976

Involvement of the glucose enzymes II of the sugar phosphotransferase system in the regulation of adenylate cyclase by glucose in Escherichia coli.

The nature of the interaction of glucose with toluene-treated cells of Escherichia coli leading to inhibition of adenylate cyclase was examined by the use of analogues. Those analogues with variations of the substituents about carbon atoms 1 or 2 (e.g. alpha-methylglucoside or 2-deoxyglucose) are inhibitory, and they are also substrates of the phosphoenolpyruvate-dependent sugar phosphotransferase system. Analogues with changes in other parts of the molecule (e.g. 3-O-methylglucose or galactose), L-glucose and several disaccharides and pentoses, do not inhibit adenylate cyclase and are not substrates of the phosphotransferase system. This correlation suggests some functional relationship between the adenylate cyclase and phosphotransferase systems. Further studies were done with mutants defective in glucose enzymes II of the phosphotransferase system (designated GPT and MPT); these two activities are measured by phosphorylation of alpha-methyl-glucoside and 2-deoxyglucose, respectively. The wild-type parent phosphorylates both analogues, and both inhibit adenylate cyclase. In the GPT- mutant, alpha-methylglucoside does not inhibit adenylate cyclase and is not phosphorylated, while 2-deoxyglucose is inhibitory and phosphorylated. In the GPT- MPT- double mutant, adenylate cyclase activity is present, but neither alpha-methylglucoside nor 2-deoxyglucose inhibits adenylate cyclase, and neither sugar is phosphorylated. These studies demonstrate that glucose inhibition of adenylate cyclase in toluene-treated cells requires an interaction of this sugar with either the GPT or mpt enzyme II of the phosphotransferase system.

Adenylyl Cyclases↗

PubMed1976

Regulation of carbohydrate uptake and adenylate cyclase activity mediated by the enzymes II of the phosphoenolpyruvate: sugar phosphotransferase system in Escherichia coli.

The uptake of various carbohydrates and the synthesis of adenosine 3':5'-monophosphate (cyclic AMP) are subject to inhibition by sugar substrates of the phosphoenolpyruvate:sugar phosphotransferase system in Escherichia coli. The induced synthesis of the sugar-specific components of the phosphotransferase system was studied and correlated with the induction of regulatory interactions controlling glycerol uptake and net cyclic AMP synthesis. Activities of the Enzyme II complexes specific for glucose, fructose, and mannitol were measured both in vivo and in vitro. These activities were induced 8- to 40-fold by growth in the presence of the appropriate substrate-inducers. Cross inducer specificities were noted. Maximal inhibition of glycerol uptake and cyclic AMP synthesis by a sugar substrate of the phosphotransferase system required induction of the Enzyme II complex specific for that sugar and was abolished by mutations which destroyed Enzyme II activity. The inducer specificities of the regulatory systems and of the Enzymes II were found to be the same. A mutation which depressed the cellular activity of Enzyme I of the phosphotransferase system did not relieve sensitivity to inhibition. The results suggest that adenylate cyclase and several carbohydrate permeases are subject to coordinate regulation by a mechanism which depends on the catalytic activities of the protein components of the phosphotransferase system.

Adenylyl Cyclases↗

PubMed1977

[Some properties of cytoplasmic thymidine kinase and nucleoside phosphotransferase from rat liver].

The activities of two deoxythymidine-phosphorylating enzymes--thymidine kinase and nucleoside phosphotransferase--were found in the cytoplasmic fraction of normal and regenerating rat liver. The specific activity of nucleoside phosphotransferase appeared to be by 50% higher than that of thymidine kinase. Nucleoside phosphotransferase has a broad specificity for the phosphate donor. This enzyme is more stable to heating and prolonged dialysis as compared to thymidine kinase. The enzymes respond differently to the addition of d-TTP, d-CTP and sturins A and B: thymidine kinase is strongly inhibited by these agents whereas nucleoside phosphotransferase is insensitive to d-TTP and d-CTP and is only slightly inhibited by sturins. On the other hand the activity of nucleoside phosphotransferase is considerably decreased after addition of ATP. Changes in the activities of both enzymes during 50 hrs following partial hepatectomy were studied. Two activity maxima were observed at 20-22 and 40-46 hrs of regeneration. Using polyacrylamide gel electrophoresis, three isoforms of both enzymes were found. The ratio between the isoenzyme content of the two enzymes from the cytoplasmic fraction of regenerating liver varied as compared to normal.

Animals↗

PubMed1976

Protein phosphotransferase activities and cyclic nucleotide action in proliferating lymphocytes.

Cyclic nucleotide levels, protein phosphotransferase activities, and cyclic nucleotide-binding proteins have been determined and partially characterized in the mouse lymphosarcoma P1798. This system is used as a model to understand the function of these activities in a rapidly proliferating cell. Adenosine 3':5'-monophosphate (cAMP) concentrations are 5-fold higher in the lymphosarcoma cells than in thymocytes. In both the thymocytes and malignant tissue, cAMP concentrations are increased by physiological concentrations of epinephrine and prostaglandin. The guanosine 3':5'-monophosphate (cGMP) level in the lymphosarcoma is 0.1 pmole/10(6) cells and is not modified by acetylcholine, prostaglandin F2alpha, or concanavalin A. Four protein phosphotransferase activities have been identified in the lymphosarcoma. These are the cAMP-dependent protein kinase type I and II isozymes and a "histone kinase" and a "phosvitin kinase"; neither of the latter two is regulated by cyclic nucleotides. Characterization of these enzymes was based on fractionation by DE 52 chromatography, substrate specificity, interaction with the protein inhibitor of cAMP-dependent protein kinases, and sucrose gradient sedimentation rates. Both the cAMP-dependent protein phosphotransferase activity and the phosvitin phosphotransferase activity are 2-to 4-fold elevated in the lymphosarcoma cells in comparison to thymocytes. cAMP binding is associated with both the type I and II isozymes and with a fraction tentatively designated as the regulatory subunit of these enzymes. cGMP also binds to this later fraction and to the partially purified fraction containing the type IcAMP-dependent enzyme. The histone phosphotransferase activity of this fraction is also stimulated by cGMP, but studies of the number of binding sites and of absorption to cAMP and cGMP affinity resins indicated that this fraction contains more than one species of cyclic nucleotide-binding protein.

Animals↗

PubMed1977

Unmasking of an essential thiol during function of the membrane bound enzyme II of the phosphoenolpyruvate glucose phosphotransferase system of Escherichia coli.

The addition of N-ethylmaleimide (MalNEt), or of fluoro dinitrobenzene to a suspension of Escherichia coli during the phosphorylating uptake of methyl-alpha-D-glucopyranoside (Me-Glc), a glucose analog, stops uptake and phosphorylation and causes the loss of previously accumulated sugar and of its phosphate ester. After removal of the reagents, the phosphotransferase system remains irreversibly inactive. Pretreatment of the bacteria with the same reagents under the same conditions of concentration, pH, temperature and for the same length of time causes very little inactivation. Mercuric chloride, a reversible inactivator, prevents the phosphotransferase system from reacting simultaneously with MaINEt or with fluorodinitrobenzene. This protection strongly suggests that all three reagents react with the same site, presumably an -SH group. The change which makes this site available to the reagents depends on the phosphorylative uptake of Me-Glc. Preload of the cells and efflux of Me-Glc do not achieve the same change. The rate of inactivation is directly proportional to the rate of phosphorylative uptake. When the Km of phosphorylative uptake is modified by an uncoupling agent, the substrate concentration allowing half maximal rate of inactivation by MaINEt changes accordingly. The reactive sites of the phosphotransferase system can also be made accessible to the -SH group reagents by fluoride inhibition of phosphoenolpyruvate synthesis. This suggests that the inactivator resistent form is an "energized form" of the enzyme. The unmasking of the reactive site is not due to a change in transmembrane penetration of the reagents since incubation of toluene treated cells with MaINEt in the presence of phosphoenolpyruvate fails to inactivate the phosphotransferase activity, while incubation with MaINEt plus Me-Glc causes fast inactivation.

Binding Sites↗