Directed Evolution of Aminoglycoside Phosphotransferase (3′) Type IIIa Variants That Inactivate Amikacin but Impose Significant Fitness Costs

The rules that govern adaptive protein evolution remain incompletely understood. Aminoglycoside aminotransferase (3′) type IIIa (hereafter abbreviated APH(3′)-IIIa) is a good model enzyme because it inactivates kanamycin efficiently; it recognizes other aminoglycoside antibiotics, including amikacin, but not nearly as well. Here we direct the evolution of APH(3′)-IIIa variants with increased activity against amikacin. After four rounds of random mutation and selection in Escherichia coli, the minimum inhibitory concentration of amikacin rose from 18 micrograms/mL (wild-type enzyme) to over 1200 micrograms/mL (clone 4.1). The artificially evolved 4.1 APH(3′)-IIIa variant exhibited 19-fold greater catalytic efficiency (k _cat/K _M) than did the wild-type enzyme in reactions with amikacin. E. coli expressing the evolved 4.1 APH(3′)-IIIa also exhibited a four-fold decrease in fitness (as measured by counting colony forming units in liquid cultures with the same optical density) compared with isogenic cells expressing the wild-type protein under non-selective conditions. We speculate that these fitness costs, in combination with the prevalence of other amikacin-modifying enzymes, hinder the evolution of APH(3′)-IIIa in clinical settings.     

Control of 5′,5′-Dinucleoside Triphosphate Catabolism by APH1, a Saccharomyces cerevisiae Analog of Human FHIT

The putative human tumor suppressor gene FHIT (fragile histidine triad) (M. Ohta et al., Cell 84:587–597, 1996) encodes a protein behaving in vitro as a dinucleoside 5′,5′′′-P¹,P³-triphosphate (Ap₃A) hydrolase. In this report, we show that the Saccharomyces cerevisiae APH1 gene product, which resembles human Fhit protein, also hydrolyzes dinucleoside 5′,5′-polyphosphates, with Ap₃A being the preferred substrate. Accordingly, disruption of the APH1 gene produced viable S. cerevisiae cells containing reduced Ap₃A-hydrolyzing activity and a 30-fold-elevated Ap₃N concentration.     

Adenosine triphosphatase in isolated membranes of Staphylococcus aureus

The preparation of cytoplasmic membranes from suspensions of Staphylococcus aureus lysed by an enzyme recently isolated in these laboratories is described. These membranes contained: protein, 34.4%; ribonucleic acid, 6.6%; lipids, 34.5%; and total phosphorus, 1.4%. Such membranes exhibited adenosine 5′-triphosphatase (E.C. 3.6.1.3) activity, liberating orthophosphate at an initial rate of 0.53 μmole per min per mg of protein under optimal conditions. The enzyme was Mg⁺⁺-dependent and K⁺- or Na⁺-stimulated. Maximal activity was observed with a molar adenosine 5′-triphosphate (ATP) to Mg⁺⁺ ratio of 1. One mole of orthophosphate was liberated per mole of ATP; the other product of digestion was adenosine 5′-diphosphate. Inorganic pyrophosphate and the 5′-triphosphates of guanosine, uridine, and cytidine were also attacked by membrane preparations, but more slowly than ATP. Ouabain, p-chloromercuribenzoate, and 2,4-dinitrophenol did not alter adenosine triphosphatase activity, whereas both Atebrine and chlorpromazine were inhibitory.     

Purification and Regulatory Properties of Fructose 1,6-Diphosphatase from Hydrogenomonas eutropha

Fructose diphosphatase of Hydrogenomonas eutropha H 16, produced during autotrophic growth, was purified 247-fold from extracts of cells. The molecular weight of the enzyme was estimated to be 170,000. The enzyme showed a pH optimum of 8.5 in both crude extracts and purified preparation. The shape of the pH curve was not changed in the presence of ethylenediaminetetraacetic acid. The enzyme required Mg²⁺ for activity. The MgCl₂ saturation curve was sigmoidal and the degree of positive cooperativity increased at lower fructose diphosphate concentrations. Mn²⁺ can replace Mg²⁺, but maximal activity was lower than that observed with Mg²⁺ and the optimal concentration range was narrow. The fructose diphosphate curve was also sigmoidal. The purified enzyme also hydrolyzed sedoheptulose diphosphate but at a much lower rate than fructose diphosphate. The enzyme was not inhibited by adenosine 5′-monophosphate but was inhibited by ribulose 5-phosphate and adenosine 5′-triphosphate. Adenosine 5′-triphosphate did not affect the degree of cooperativity among the sites for fructose diphosphate. The inhibition by adenosine 5′-triphosphate was mixed and by ribulose 5-phosphate was noncompetitive. An attempt was made to correlate the properties of fructose diphosphatase from H. eutropha with its physiological role during autotrophic growth.     

Isolation of uridine 5'-pyrophosphate glucuronic Acid pyrophosphorylase and its assay using p-pyrophosphate

A procedure was devised to detect and assay uridine 5′-pyrophosphate (UDP)-glucuronic acid pyrophosphorylase in plant extracts. Substrates are UDP-glucuronic acid and ³²P-pyrophosphate, and the ³²P-uridine 5′-triphosphate produced is selectively adsorbed to charcoal. The charcoal adsorption procedure is a modification of that used to determine ³²P-adenosine 5′-triphosphate produced by adenosine 5′-pyrophosphate glucose pyrophosphorylase, and the modification greatly improves the retention of uridine 5′-triphosphate.     

The preparation of adenosine 5′-pyrophosphate by a non-enzymic method

1. A non-enzymic method for the preparation of adenosine 5′-diphosphate is described, in which the terminal phosphate of adenosine 5′-triphosphate is transferred to methanol in the presence of hydrochloric acid. The final purified product can be obtained in 60% yield. 2. Experiments with [¹⁴C]methanol showed that no methylation of the adenosine diphosphate occurs during the reaction. 3. Confirmation that the pyrophosphate moiety of the adenosine diphosphate produced was in the 5′-position was obtained by: (a) periodate oxidation; (b) treatment with apyrase and examination of the resulting adenylic acid isomer by paper chromatography. 4. The method appears to be generally applicable to the preparation of nucleoside 5′-diphosphates from the corresponding nucleoside 5′-triphosphates.     

Turnip Yellow Mosaic Virus RNA as a Substrate of the Transfer RNA Nucleotidyltransferase II. Incorporation of Cytidine 5'-Monophosphate and Determination of a Short Nucleotide Sequence at the 3' End of the RNA

Turnip yellow mosaic virus (TYMV) RNA treated with snake venom phosphodiesterase accepts cytidine 5′-monophosphate and adenosine 5′-monophosphate (AMP) when it is incubated in the presence of cytidine 5′-triphosphate (CTP), adenosine 5′-triphosphate, and Escherichia coli transfer RNA nucleotidyltransferase; untreated TYMV RNA accepts only AMP. When α ³²PCTP was used for terminal labeling, the nearest neighbor analyses and the anallyses after action of various nucleases showed that the sequence of five nucleotides at the 3′ end of TYMV RNA is: pGpCpApCpC. A nuclease present in commerical preparations of snake venom phosphodiesterase leads to the fragmentation of TYMV RNA, the 3′ end of which is found in a fragment having a sedimentation constant close to 5s.     

Comparison of Methods for Extraction of Bacterial Adenine Nucleotides Determined by Firefly Assay

Adenine nucleotides in Escherichia coli, Bacillus cereus, Klebsiella pneumoniae, Staphylococcus aureus, and Pseudomonas aeruginosa were extracted using 10 different methods. Extracts were assayed for adenosine 5′-triphosphate (ATP), adenosine 5′-diphosphate (ADP), and adenosine 5′-monophosphate (AMP) by the firefly method using an improved procedure. Analytical interference by bacterial enzymes not inactivated during the extraction was found to be a major problem. However, these enzymes were inactivated to a considerable extent by the inclusion of ethylenediaminetetraacetate in the extraction reagent. The 10 extraction methods were compared with respect to yield of adenine nucleotides, interference with the enzymic assay, reproducibility of the method, and stability of the extracts. Results indicated that extraction with trichloroacetic acid was the method most closely reflecting actual levels of ATP, ADP, and AMP in intact bacterial cells. However, for the extraction of ATP in some bacterial strains several other methods may be used and may be advantageous from a practical point of view.     

Further studies on the bicarbonate stimulation of photophosphorylation in isolated chloroplasts

The bicarbonate effect in stimulating the rate of photophosphorylation by isolated spinach (Spinacia oleracea var. Virginia blight-resistant savoy) chloroplasts at a pH below the optimum has been re-examined. Its seasonal nature may be related to the hormonal status of the plants. Bicarbonate anions stimulate adenosine 5′-triphosphate synthesis if added in the final, adenosine 5′-triphosphate-forming stage of either a postillumination or an acid-base experiment. They also stimulate the membrane-bound, Mg²⁺-dependent adenosine 5′-triphosphatase of chloroplasts, and the Ca²⁺-dependent adenosine 5′-triphosphatase of detached coupling factor. These and other data point to the interaction between energized thylakoid membranes and the coupling factor as the probable site of action of bicarbonate anions when they stimulate photophosphorylation.     

Effect of Glucose and Adenosine Phosphates on Production of Extracellular Carbohydrases of Alternaria solani

Production of carbohydrases by Alternaria solani is inhibited by glucose under low growth conditions. In an enriched medium, glucose has little effect on the production of polygalacturonase and cellulase while it still suppresses production of β-glucosidase. Low levels of all three enzymes were produced in the absence of their respective substrates. Such regulation has been found with many organisms. However, far greater production of these carbohydrases occurred with additions of adenosine phosphates to the growth media. Highest stimulation of enzyme production was by adenosine 5′-phosphate. Adenosine 5′-triphosphate and cyclic 3′, 5′-adenosine monophosphate gave lesser amounts. Starvation appears to induce production of extracellular carbohydrases and adenosine 5′-phosphate may have a role in the starvation process.     

Revisiting the Nucleotide and Aminoglycoside Substrate Specificity of the Bifunctional Aminoglycoside Acetyltransferase(6′)-Ie/Aminoglycoside Phosphotransferase(2″)-Ia Enzyme

The bifunctional aminoglycoside-modifying enzyme aminoglycoside acetyltransferase(6′)-Ie/aminoglycoside phosphotransferase(2″)-Ia, or AAC(6′)-Ie/APH(2″)-Ia, is the major source of aminoglycoside resistance in Gram-positive bacterial pathogens. In previous studies, using ATP as the cosubstrate, it was reported that the APH(2″)-Ia domain of this enzyme is unique among aminoglycoside phosphotransferases, having the ability to inactivate an unusually broad spectrum of aminoglycosides, including 4,6- and 4,5-disubstituted and atypical. We recently demonstrated that GTP, and not ATP, is the preferred cosubstrate of this enzyme. We now show, using competition assays between ATP and GTP, that GTP is the exclusive phosphate donor at intracellular nucleotide levels. In light of these findings, we reevaluated the substrate profile of the phosphotransferase domain of this clinically important enzyme. Steady-state kinetic characterization using the phosphate donor GTP demonstrates that AAC(6′)-Ie/APH(2″)-Ia phosphorylates 4,6-disubstituted aminoglycosides with high efficiency (k_cat/K_m = 10⁵-10⁷ m⁻¹ s⁻¹). Despite this proficiency, no resistance is conferred to some of these antibiotics by the enzyme in vivo. We now show that phosphorylation of 4,5-disubstituted and atypical aminoglycosides are negligible and thus these antibiotics are not substrates. Instead, these aminoglycosides tend to stimulate an intrinsic GTPase activity of the enzyme. Taken together, our data show that the bifunctional enzyme efficiently phosphorylates only 4,6-disubstituted antibiotics; however, phosphorylation does not necessarily result in bacterial resistance. Hence, the APH(2″)-Ia domain of the bifunctional AAC(6′)-Ie/APH(2″)-Ia enzyme is a bona fide GTP-dependent kinase with a narrow substrate profile, including only 4,6-disubstituted aminoglycosides.     

The Crystal Structures of Substrate and Nucleotide Complexes of Enterococcus faecium Aminoglycoside-2′′-Phosphotransferase-IIa [APH(2′′)-IIa] Provide Insights into Substrate Selectivity in the APH(2′′) Subfamily

Aminoglycoside-2′′-phosphotransferase-IIa [APH(2′′)-IIa] is one of a number of homologous bacterial enzymes responsible for the deactivation of the aminoglycoside family of antibiotics and is thus a major component in bacterial resistance to these compounds. APH(2′′)-IIa produces resistance to several clinically important aminoglycosides (including kanamycin and gentamicin) in both gram-positive and gram-negative bacteria, most notably in Enterococcus species. We have determined the structures of two complexes of APH(2′′)-IIa, the binary gentamicin complex and a ternary complex containing adenosine-5′-(β,γ-methylene)triphosphate (AMPPCP) and streptomycin. This is the first crystal structure of a member of the APH(2′′) family of aminoglycoside phosphotransferases. The structure of the gentamicin-APH(2′′)-IIa complex was solved by multiwavelength anomalous diffraction methods from a single selenomethionine-substituted crystal and was refined to a crystallographic R factor of 0.210 (R_free, 0.271) at a resolution of 2.5 Å. The structure of the AMPPCP-streptomycin complex was solved by molecular replacement using the gentamicin-APH(2′′)-IIa complex as the starting model. The enzyme has a two-domain structure with the substrate binding site located in a cleft in the C-terminal domain. Gentamicin binding is facilitated by a number of conserved acidic residues lining the binding cleft, with the A and B rings of the substrate forming the majority of the interactions. The inhibitor streptomycin, although binding in the same pocket as gentamicin, is orientated such that no potential phosphorylation sites are adjacent to the catalytic aspartate residue. The binding of gentamicin and streptomycin provides structural insights into the substrate selectivity of the APH(2′′) subfamily of aminoglycoside phosphotransferases, specifically, the selectivity between the 4,6-disubstituted and the 4,5-disubstituted aminoglycosides.The emergence of bacteria resistant to several important classes of antibiotics has become a major clinical problem over the last few years. Almost every antibacterial compound in clinical use today has associated examples of resistant bacterial isolates (39), including life-threatening strains of Escherichia coli, Mycobacterium tuberculosis, Pseudomonas aeruginosa, and various enterococci. The latter are among the most common antibiotic-resistance bacteria isolated from patients with nosocomial infections in the United States today. The synergistic use of either ampicillin or vancomycin with an aminoglycoside, such as kanamycin or gentamicin, has long been the optimal therapy for serious enterococcal infections; however, many previously susceptible enterococcal strains have since acquired resistance to the aminoglycosides. The mechanisms of resistance are many and varied, although only three are readily understood: (i) mutation of the ribosomal target, (ii) reduced permeability and/or increased efflux of the drug, and (iii) enzymatic deactivation of the drug. Resistance to the aminoglycosides through enzymatic deactivation, although seemingly straightforward, is in reality a complex problem involving three different classes of enzyme. These enzyme classes are the ATP-dependent phosphotransferases (APH) and adenyltransferases (ANT), and the acetyl coenzyme A-dependent N-acetyltransferases (AAC). This area of research has been extensively reviewed in the past few years (2, 4, 13, 29, 39, 47, 52, 53).Originally isolated from soil bacteria, including various species of Streptomyces and Micromonospora (20), the aminoglycosides are a family of potent, broad-spectrum antibiotics that includes clinically relevant drugs such as gentamicin, neomycin, amikacin, kanamycin, and streptomycin. The structures of these compounds, with the exception of that of streptomycin, are all similar, consisting of a central aminocyclitol ring (the B ring) with two or three substituted aminoglycan rings (A, C, and in some cases, D) attached at either the 4 and 5 positions (the 4,5-disubstituted aminoglycosides, which include neomycin and lividomycin) or the 4 and 6 positions (the 4,6-disubstituted aminoglycosides, such as gentamicin and kanamycin). Streptomycin, a competitive inhibitor of aminoglycoside-2′′-phosphotransferase-IIa [APH(2′′)-IIa] (45), is an atypical aminoglycoside that does not fall into either the 4,5-disubstituted or 4,6-disubstituted classes. It has a modified ribose (ring B) attached to position 4 on a 1,3-diguanidinium-substituted aminocyclitol ring (ring A) with no substituent at the 5 or 6 position. The structures of gentamicin, kanamycin, neomycin, and streptomycin are shown in Fig. ​Fig.1.1. The aminoglycosides are targeted to the 16S rRNA of the bacterial 30S ribosomal subunit, where they selectively bind to the decoding aminoacyl (A) site (31, 51) and stabilize the conformation of the tRNA bound to a cognate mRNA codon. This decreases the dissociation rate of aminoacyl-tRNA and promotes miscoding (28). The structures of a number of the aminoglycosides with either the 30S subunit or oligonucleotides containing minimal A sites are known (51).Open in a separate windowFIG. 1.Structures of gentamicin, kanamycin, streptomycin, and neomycin. Gentamicin and kanamycin are classified as 4,6-disubstituted aminoglycosides, whereas neomycin is an example of a 4,5-disubstituted compound. The three structural variants which comprise gentamicin C are indicated. Amikacin is similar to kanamycin, although the substituent on the N1 amine is a 4-amino-2-hydroxy-1-oxobutyl group. Taken together, the A and B rings of aminoglycosides, such as gentamicin, kanamycin, and neomycin, are commonly known as the neamine moiety.The enzymes which deactivate the aminoglycosides are named according to the reaction they catalyze and the site on the aminoglycoside at which they act. The APH(2′′) enzymes, which give rise to high-level resistance to gentamicin in enterococci, phosphorylate gentamicin and kanamycin at the 2′′-hydroxyl group of the C ring (Fig. ​(Fig.1).1). The APH(3′) enzymes, another major subfamily of the phosphotransferases, phosphorylate kanamycin and neomycin at the 3′-hydroxyl on the A ring but cannot deactivate gentamicin, since it has no corresponding 3′-hydroxyl. The individual members of each family can normally bind only a subset of the available drugs, and this difference in drug specificity is known as the resistance profile, designated with a roman numeral and, in some cases, a letter identifying a specific gene. The first APH(2′′) enzyme discovered for enterococci was the bifunctional AAC(6′)-Ie-APH(2′′)-Ia enzyme, which possesses both 6′-acetylating and 2′′-phosphorylating activities (17, 33). Enterococci with the corresponding gene show resistance to almost all clinically relevant aminoglycosides (38). Four additional APH(2′′) enzymes have since been isolated for Enterococcus spp.; they are designated APH(2′′)-Ib (27), APH(2′′)-Ic (11), APH(2′′)-Id (46), and APH(2′′)-Ie (10) and were initially classified as genetic variants of an APH(2′′)-I-type enzyme. Recently, APH(2′′)-Ib, APH(2′′)-Ic, and APH(2′′)-Id have been reclassified as distinct enzymes with different resistance profiles and, more importantly, different nucleotide specificities, such that they are now named APH(2′′)-IIa, APH(2′′)-IIIa, and APH(2′′)-IVa, respectively (44). APH(2′′)-Ie was not included in the latter study, but based upon the very high sequence similarity with APH(2′′)-IVa (93%) (see Table S1 in the supplemental material), it is possible that it is a genetic variant of APH(2′′)-IVa.Structural details are currently known for only two members of the APH(3′) family, APH(3′)-IIIa (5, 18, 23) and APH(3′)-IIa (37). These enzymes share a two-domain structure similar to the catalytic domains of the eukaryotic Ser/Thr and Tyr protein kinases. Moreover, the phosphotransferases and kinases share several important sequence motifs related to nucleotide binding and phosphoryl transfer, most notably the catalytic loop (HXDXXXXN) and the activation segment (GXIDXG), where X is any amino acid. Not surprisingly, the catalytic mechanisms of the phosphotransferases and the kinases are identical, involving the nucleophilic attack by the target hydroxyl on the γ phosphate of ATP, facilitated by a conserved aspartate residue from the catalytic loop (29, 54). A comparison of the known APH(2′′) and APH(3′) sequences shows that the two families of phosphotransferases share these kinase-like motifs, and there appears to be some partial conservation of acidic residues in the substrate binding region. It has been suggested that their structures may be similar (37). Here, we report the first structure of an APH(2′′) enzyme, APH(2′′)-IIa as the binary complex with the preferred substrate gentamicin and the ternary complex with the nonhydrolyzable ATP analog adenosine-5′-(β,γ-methylene)triphosphate (AMPPCP) and the competitive inhibitor streptomycin.     

Studies on the presence of adenosine cyclic 3':5'-monophosphate in oat coleoptiles

The incorporation of adenosine-8-¹⁴C into adenosine cyclic 3′:5′-monophosphate in coleoptile-first leaf segments of Avena sativa L. was investigated. Homogenates of segments incubated in adenosine-8-¹⁴C for either 4 or 10 hours were partially purified by thin layer chromatography followed by paper electrophoresis. A radioactive fraction, less than 0.06% of the ¹⁴C present in the original homogenate, migrated as adenosine cyclic 3′:5′-monophosphate during electrophoresis. Upon treatment with cyclic nucleotide phosphodiesterase, however, less than 10% of this radioactive fraction appeared as 5′-AMP. Deamination with NaNO₂ as well as further chromatographical purification also suggested that only a small fraction of the ¹⁴C in the partially purified samples could be in adenosine cyclic 3′:5′-monophosphate. The data suggest that levels of this nucleotide can probably be no greater than 7 to 11 picomoles per gram of fresh weight in oat coleoptiles. Treatment of such coleoptiles with physiologically active concentrations of indoleacetic acid, furthermore, had no significant effect on the ¹⁴C radioactivity in marker adenosine cyclic 3′:5′-monophosphate-containing fractions at any stage of purification during several experiments.     

Adenosine 5'-sulphatophosphate kinase activity in spinach leaf tissue

1. An F⁻-insensitive 3′-nucleotidase was purified from spinach leaf tissue; the enzyme hydrolysed 3′-AMP, 3′-CMP and adenosine 3′-phosphate 5′-sulphatophosphate but not adenosine 5′-nucleotides nor PP_i. The pH optimum of the enzyme was 7.5; K_m (3′-AMP) was approx. 0.8mm and K_m (3′-CMP) was approx. 3.3mm. 3′-Nucleotidase activity was not associated with chloroplasts. Purified Mg²⁺-dependent pyrophosphatase, free from F⁻-insensitive 3′-nucleotidase, catalysed some hydrolysis of 3′-AMP; this activity was F⁻-sensitive. 2. Adenosine 5′-sulphatophosphate kinase activity was demonstrated in crude spinach extracts supplied with 3′-AMP by the synthesis of the sulphate ester of 2-naphthol in the presence of purified phenol sulphotransferase; purified ATP sulphurylase and pyrophosphatase were also added to synthesize adenosine 5′-sulphatophosphate. Adenosine 5′-sulphatophosphate kinase activity was associated with chloroplasts and was released by sonication. 3. Isolated chloroplasts synthesized adenosine 3′-phosphate 5′-sulphatophosphate from sulphate and ATP in the presence of a 3′-nucleotide; the formation of adenosine 5′-sulphatophosphate was negligible. In the absence of a 3′-nucleotide the synthesis of adenosine 3′-phosphate 5′-sulphatophosphate was negligible, but the formation of adenosine 5′-sulphatophosphate was readily detected. Some properties of the synthesis of adenosine 3′-phosphate 5′-sulphatophosphate by isolated chloroplasts are described. 4. Adenosine 3′-phosphate 5′-sulphatophosphate, synthesized by isolated chloroplasts, was characterized by specific enzyme methods, electrophoresis and i.r. spectrophotometry. 5. Isolated chloroplasts catalysed the incorporation of sulphur from sulphate into cystine/cysteine; the incorporation was enhanced by 3′-AMP and l-serine. It was concluded that adenosine 3′-phosphate 5′-sulphatophosphate is an intermediate in the incorporation of sulphur from sulphate into cystine/cysteine.     

Pyruvate Kinase of Streptococcus lactis

The kinetic properties of pyruvate kinase (ATP:pyruvate-phosphotransferase, EC 2.7.1.40) from Streptococcus lactis have been investigated. Positive homotropic kinetics were observed with phosphoenolpyruvate and adenosine 5′-diphosphate, resulting in a sigmoid relationship between reaction velocity and substrate concentrations. This relationship was abolished with an excess of the heterotropic effector fructose-1,6-diphosphate, giving a typical Michaelis-Menten relationship. Increasing the concentration of fructose-1,6-diphosphate increased the apparent V_max values and decreased the K_m values for both substrates. Catalysis by pyruvate kinase proceeded optimally at pH 6.9 to 7.5 and was markedly inhibited by inorganic phosphate and sulfate ions. Under certain conditions adenosine 5′-triphosphate also caused inhibition. The K_m values for phosphoenolpyruvate and adenosine 5′-diphosphate in the presence of 2 mM fructose-1,6-diphosphate were 0.17 mM and 1 mM, respectively. The concentration of fructose-1,6-diphosphate giving one-half maximal velocity with 2 mM phosphoenolpyruvate and 5 mM adenosine 5′-diphosphate was 0.07 mM. The intracellular concentrations of these metabolites (0.8 mM phosphoenolpyruvate, 2.4 mM adenosine 5′-diphosphate, and 18 mM fructose-1,6-diphosphate) suggest that the pyruvate kinase in S. lactis approaches maximal activity in exponentially growing cells. The role of pyruvate kinase in the regulation of the glycolytic pathway in lactic streptococci is discussed.     

Identification of a 5′-Deoxyadenosine Deaminase in Methanocaldococcus jannaschii and Its Possible Role in Recycling the Radical S-Adenosylmethionine Enzyme Reaction Product 5′-Deoxyadenosine

We characterize here the MJ1541 gene product from Methanocaldococcus jannaschii, an enzyme that was annotated as a 5′-methylthioadenosine/S-adenosylhomocysteine deaminase (EC 3.5.4.31/3.5.4.28). The MJ1541 gene product catalyzes the conversion of 5′-deoxyadenosine to 5′-deoxyinosine as its major product but will also deaminate 5′-methylthioadenosine, S-adenosylhomocysteine, and adenosine to a small extent. On the basis of these findings, we are naming this new enzyme 5′-deoxyadenosine deaminase (DadD). The K_m for 5′-deoxyadenosine was found to be 14.0 ± 1.2 μM with a k_cat/K_m of 9.1 × 10⁹ M⁻¹ s⁻¹. Radical S-adenosylmethionine (SAM) enzymes account for nearly 2% of the M. jannaschii genome, where the major SAM derived products is 5′-deoxyadenosine. Since 5′-dA has been demonstrated to be an inhibitor of radical SAM enzymes; a pathway for removing this product must be present. We propose here that DadD is involved in the recycling of 5′-deoxyadenosine, whereupon the 5′-deoxyribose moiety of 5′-deoxyinosine is further metabolized to deoxyhexoses used for the biosynthesis of aromatic amino acids in methanogens.     

Preparation of Cells Permeable to Macromolecules by Treatment with Toluene: Studies of Transfer Ribonucleic Acid Nucleotidyltransferase

Transfer ribonucleic acid (tRNA) nucleotidyltransferase was studied after making cells permeable to macromolecules by treatment with toluene. The conditions of toluene treatment necessary for obtaining maximal activity were defined. Toluene treatment was most efficient when carried out for 5 min at 37 C at pH 9.0 on log-phase cells. No activity could be detected if cells were treated at 0 C, or in the presence of MgCl₂, or if the cells were in the stationary phase of growth. However, inclusion of lysozyme and ethylenediaminetetraacetic acid during the toluene treatment did render stationary phase cells permeable. The properties of tRNA nucleotidyltransferase from toluene-treated cells were essentially identical to those of purified enzyme with regard to pH optimum, specificity for nucleoside triphosphates and tRNA, and apparent K_m values for substrates. In addition to tRNA nucleotidyltransferase, a variety of other enzymes which incorporate adenosine 5′-triphosphate into acid-precipitable material could also be detected in toluene-treated cells. Centrifugation of cells treated with toluene revealed that tRNA nucleotidyltransferase leaked out of cells, whereas other activities remained associated with the cell pellets. Chromatography of the material extracted from toluene-treated cells on Sephadex G-100 indicated that toluene treatment selectively extracts lower molecular weight proteins. The usefulness of such a procedure as an initial step in purification of such enzymes, and its application to tRNA nucleotidyltransferase, is discussed.     

A novel mechanism of selectivity against AZT by the human mitochondrial DNA polymerase

Native nucleotides show a hyperbolic concentration dependence of the pre-steady-state rate of incorporation while maintaining concentration-independent amplitude due to fast, largely irreversible pyrophosphate release. The kinetics of 3′-azido-2′,3′-dideoxythymidine (AZT) incorporation exhibit an increase in amplitude and a decrease in rate as a function of nucleotide concentration, implying that pyrophosphate release must be slow so that nucleotide binding and incorporation are thermodynamically linked. Here we develop assays to measure pyrophosphate release and show that it is fast following incorporation of thymidine 5′-triphosphate (TTP). However, pyrophosphate release is slow (0.0009 s⁻¹) after incorporation of AZT. Modeling of the complex kinetics resolves nucleotide binding (230 µM) and chemistry forward and reverse reactions, 0.38 and 0.22 s⁻¹, respectively. This unique mechanism increases selectivity against AZT incorporation by allowing reversal of the reaction and release of substrate, thereby reducing k_cat/K_m (7 × 10⁻⁶ μ M⁻¹ s⁻¹). Other azido-nucleotides (AZG, AZC and AZA) and 8-oxo-7,8-dihydroguanosine-5′-triphosphate (8-oxo-dGTP) show this same phenomena.     

Formation of adenosine 5'-phosphorofluoridate and the assay of adenyl cyclase in barley seeds

Barley seed (Hordeum vulgare L.) homogenates contain an apparent enzymatic activity which catalyzes the synthesis of adenosine 5′-phosphorofluoridate from magnesium-adenosine 5′-triphosphate and sodium fluoride. Formation of this compound may interfere with some adenyl cyclase assays which use fluoride as a component of the incubation medium. Neither adenyl cyclase activity nor endogenous adenosine 3′: 5′-monophosphate was detected in barley seed homogenates or extracts.     

Fructokinase (Fraction III) of Pea Seeds

A second fructokinase (EC 2.7.1.4) was obtained from pea seed (Pisum sativum L. var. Progress No. 9) extracts. The enzyme, termed fructokinase (fraction III), was specific for fructose and had little activity with glucose. With fructose concentrations above 0.25 millimolar, there was strong substrate inhibition at the optimum pH (8.0) and also at pH 6.6. The apparent K_m values at pH 8.0 for fructose and glucose were 0.06 millimolar and 0.14 millimolar, respectively. The apparent K_m for Mg adenosine 5′-triphosphate (MgATP) was 0.06 millimolar and excess MgATP was inhibitory. Mg²⁺ was essential for activity but the enzyme was inhibited by excess Mg²⁺ or ATP. Mg adenosine 5′-pyrophosphate was also inhibitory. Activity was stimulated by the addition of monovalent cations: of those tested K⁺, Rb⁺, and NH₄⁺ were the most effective. The possible role of fructokinase (fraction III) is discussed.