Two forms of phosphoinositol kinase from germinating mung bean seeds

Phosphoinositol kinase, the key enzyme responsible for the biosynthesis of higher inositol phosphates has been isolated from the cotyledons of mung beans germinated for 24 hr and has been resolved into two different forms, phosphoinositol kinase A and phosphoinositol kinase B. Both forms were purified to homogeneity and characterized. The K_m values for ATP with phosphoinositol kinase A (1.78 × 10^?4 M) and phosphoinositol kinase B (3.12 × 10 ^?5 M) showed that phosphoinositol kinase B had a greater affinity for ATP. ATP could be partially replaced as phosphate donor by UTP and phosphoenolpyruvate in the case of phosphoinositol kinase A but not in the case of phosphoinositol kinase B.     

4-Coumarate:CoA ligase from cell suspension cultures of Petroselinum hortense Hoffm: Partial purification,substrate specificity,and further properties

4-Coumarate:CoA ligase (EC 6.2.1.12) was isolated from 8-day-old cell suspension cultures of parsley (Petroselinum hortense Hoffm.) which had been irradiated with ultraviolet light for 15 h. The enzyme was partially purified by fractionation with MnCl₂ and (NH₄)₂SO₄ and by column chromatography on diethylaminoethyl cellulose, hydroxyapatite, and aminohexyl-Sepharose. A 90-fold increase in specific activity with an overall yield of 20% was achieved. Analytical gel electrophoresis indicated the occurrence of only one 4-coumarate:CoA ligase species in the final enzyme preparation. The enzyme was largely specific for 4-coumarate and other derivatives of cinnamic acid. 4-Coumarate had the lowest apparent K_m and the highest $\text{V}\text{K}{}_{\mathrm{m}}$ values (1.4 × 10^?5, m and 14.7 × 10⁵ pkatal × m^?1, respectively) of all substrates tested. Only the trans isomer of 4-coumarate was activated. The two cosubstrates, ATP and CoA, exhibited sigmoidal saturation kinetics, which were interpreted as indicating homotropic, allo-steric effects. A molecular weight of about 67,000 was estimated for 4-coumarate:CoA ligase. The substrate specificity of the enzyme was in agreement with its proposed function in flavonoid biosynthesis.     

Uridine-cytidine kinase. Kinetic studies and reaction mechanism.

The initial velocity pattern has been determined for uridine-cytidine kinase purified from the murine mast cell neoplasm P815. With either uridine or cytidine as phosphate acceptor, and ATP as phosphate donor, the pattern observed was one of intersecting lines, ruling out a ping-pong reaction mechanism, and suggesting that the reaction probably proceeds by the sequential addition of both substrates to the enzyme to form a ternary complex, followed by the sequential release of the two products. This pattern was obtained whether the reaction was run in 0.01 m potassium phosphate buffer, pH 7.5, or in 0.1 m Tris-HCl, pH 7.2. When analyzed by the Sequen computer program, the data indicated an apparent K_m of the enzyme for uridine of 1.5 × 10^?4m, an apparent K_m for cytidine of 4.5 × 10^?5m, and a K_m for ATP, with uridine or cytidine as phosphate acceptor, of 3.6 × 10^?3m or 2.1 × 10^?3m, respectively. The V was 1.83 μmol phosphorylated/min/mg enzyme protein for the uridine kinase reaction and 0.91 μmol for the cytidine kinase reaction.     

Phosphofructokinase of Solanum tuberosum tuber

Potato tuber phosphofructokinase was purified 19·.6-fold by a combination of ethanol fractionation and DEAE-cellulose column chromatography. The enzyme was very unstable; its pH optimum was 8·0. K_m for fructose-6-phosphate, ATP and Mg²⁺ was 2·1 × 10^?4 M, 4·5 × 10^?5 M and 4·0 × 10^?4 M respectively. ITP, GTP, UTP and CTP can act as phosphate donors, but are less active than ATP. Inhibition of enzyme activity by high levels of ATP was reversed by increasing the concentration of fructose-6-phosphate; the affinity of enzyme for fructose-6-phosphate decreased with increasing concentration of ATP. 5′-AMP, 3′,5′-AMP, 3′-AMP, deoxy AMP, UMP, IMP, CMP, GMP, ADP, CDP, GDP and UDP did not reverse the inhibition of enzyme by ATP. ADP, phosphoenolpyruvate and citrate inhibited phosphofructokinase activity but Pi did not affect it. Phosphofructokinase was not reactivated reversibly by mild change of pH and addition of effectors.     

Adenylate kinase bound to the envelope membranes of spinach chloroplasts.

The activity of adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) in both the forward (2ADP → ATP + AMP) and backward (ATP + AMP → 2ADP) reactions was found to be associated with the envelope membranes which were isolated from spinach chloroplasts. Sonication and repeated washing in a medium of high ionic strength were unable to release the enzymes from the envelope membranes. Adenylate kinase bound to the envelope is stable in the cold and inactivated by heat and acid treatments. The enzyme requires magnesium ion as an activator. The pH-activity profile of the forward reaction catalyzed by membrane-bound adenylate kinase gave a maximal activity at pH 8.5. The apparent Michaelis constant, K_m, value for ADP in the forward reaction was estimated to be 1.3 ± 0.2 × 10^?4m. A Lineweaver-Burk plot of the forward reaction gave a straight line when the reciprocal of the reaction rate was plotted versus the reciprocal, and not the square of the reciprocal, of the concentration of substrate ADP. This favors the view that the adenylate kinase bound to the chloroplast envelope has a single or equivalent binding site of Mg-ADP^?. The probable involvement of adenylate kinase bound to the chloroplast envelope in controlling the energy pool and adenylate translocation in chloroplasts is suggested.     

Purifications and properties of L-mandelate- 4-hydroxylase from Pseudomonas convexa.

An inducible l-mandelate-4-hydroxylase has been partially purified from crude extracts of Pseudomonas convexa. This enzyme catalyzed the hydroxylation of l-mandelic acid to 4-hydroxymandelic acid. It required tetrahydropteridine, NADPH, Fe²⁺, and O₂ for its activity. The approximate molecular weight of the enzyme was assessed as 91,000 by gel filtration on Sephadex G-150. The enzyme was optimally active at pH 5.4 and 38 °C. A classical Michaelis-Menten kinetic pattern was observed with l-mandelate, NADPH, and ferrous sulfate and K_m values for these substrates were found to be 1 × 10^?4, 1.9 × 10^?4, and 4.7 × 10^?5m, respectively. The enzyme is very specific for l-mandelate as substrate. Thiol inhibitors inhibited the enzyme reaction, indicating that the sulfhydryl groups may be essential for the enzyme action. Treatment of the partially purified enzyme with denaturing agents inactivated the enzyme.     

Ecto-protein kinase activity of fibroblasts.

Isolated, intact dermal fibroblasts can transfer the terminal phosphate of adenosine triphosphate, [γ-³²P]ATP, to an exogenously added macromolecule (histone). The incorporation of labeled phosphate to histone is attributed to an extracellularly directed protein kinase activity (ecto-kinase) which cannot be accounted for by soluble cytoplasmic protein kinase that might have been released and become bound to cell membranes during the cell preparation. The addition of soluble cytoplasmic enzyme preparations to the cell suspension was fully recoverable in the supernatant and the first wash. The activity of ectokinase was abolished by incubation of intact cells with trypsin for 5 min, whereas the activity of cytoplasmic enzyme was unaffected by the trypsin treatment. These data suggest that dermal fibroblasts contain protein kinase on the outer surface of plasma membrane which can phosphorylate exogenously added macromolecules. The ecto-protein kinase activity is dependent on cell number, time of incubation, and the concentration of Mg²⁺ in the reaction mixture. Lineweaver-Burk plot analyses yielded K_m values for ATP and histone of 7 × 10^?5 and 3 × 10^?6m, respectively. The ecto-protein kinase activity of normal fibroblasts and fibrosarcoma cells were also compared. The enzyme activity of normal cells was higher than that of the malignant cells and was not significantly affected by cyclic nucleotides, whereas the activity of the malignant cells were stimulated by the addition of micromolar concentrations of the cyclic nucleotides.     

Further Characterization of Ureido Ring Synthetase from Pseudomonas graveolens

Detailed enzymatic properties of the ureido ring synthetase purified from Pseudomonas graveolens were investigated. Nucleotide specificity studies indicated that CTP, UTP, GTP, and ITP were each tenth to one-fifth as active as ATP. The effect of substrate concentration was examined. The Km values for 7,8-diaminopelargonic acid, biotin diaminocarboxylic acid, NaHCO₃, ATP, and MgCl₂ were 1 × 10^?4 M, 4 × 10^?5 M, 1 × 10^?2 m, 5 × 10^?5 M, and 3 × 10^?3 M, respectively. It was elucidated that only ADP was produced from ATP in both the reaction of desthiobiotin synthesis from 7,8-diaminopelargonic acid and biotin synthesis from biotin diaminocarboxylic acid. The reaction was remarkably inhibited by Ni²⁺, Cd²⁺, Cu²⁺, Ag⁺, and As³⁺, while Mn²⁺ remarkably enhanced the enzyme reaction. The reaction was remarkably inhibited by metal-chelating reagents. It was elucidated that ADP had a competitively inhibiting effect on this enzyme reaction. 7,8-DiaminopeIargonic acid, which is the substrate for the desthiobiotin synthesis, competitively inhibited the biotin synthesis from biotin diaminocarboxylic acid. The stoichiometry of the desthiobiotin synthesis indicated that the formation ratio of desthiobiotin to ADP was 1 to 1.     

Lunularic acid decarboxylase from the liverwort Conocephalum conicum

Some properties of a preparation of an enzyme, lunularic acid decarboxylase, from the liverwort Conocephalum conicum are described. The enzyme is normally bound and could be solubilized with Triton X-100; at least some of the bound decarboxylase activity appears to be associated with chloroplasts. For lunularic acid the enzyme has K_m 8.7 × 10^?5 M (pH 7.8 and 30°). Some substrate analogues have been tested but no other substrate was found. Pinosylvic acid is a competitive inhibitor for the enzyme, K_i 1.2 × 10^?4 M (pH 7.8 and 30°). No product inhibition was observed. Lunularic acid decarboxylase activity has also been observed with a cell-free system from Lunularia cruciata.     

Properties of enzyme activities involved in protein phosphorylatino-dephosphorylation of the thyroid plasma membranes

Bovine thyroid tissue exhibited cAMP-dependent and Ca²⁺-dependent protien kinase activities as well as a basal (cAMP- and Ca²⁺-independent) one, and phosphoprotein phosphatase activity. Although the former two protein kiniase activities were not clearly demonstrated using endogenous protein as substrate, they were clearly shown in soluble, particulate and plasma membrane fractions using exogenous histones as substrate. The highest specific activities were in the plasma membrane. The apparent K_m values of cAMP and Ca²⁺ for the membrane-bound protein kinase were 5·10^?8 M and 8.3·10^?4M (in the presence of 1 mM EGTA), respectively. The apparent K_m values of Mg²⁺ were 7·10^?4 M (without cAMP and Ca²⁺, 5·10^?4 M (with cAMP) and 1.3·10^?3 M (with Ca²⁺), and those ATP were 3.5·10^?5 M (with or without cAMP) and 8.5·10^?5 M (with Ca²⁺). The Ca²⁺-dependent protein kinase could be dissociated from the membrane by EGTA-washing. The enzyme activity so released was further activated by added phospholipid (phosphatidylserine/1,3-diolein), but not by calmodulin. Phosphoprotein phosphatase activity was also clearly demonstrated in all of the fractions using ³²P-labeled mixed histones as substrate. The activity was not modified by either cAMP or Ca²⁺, but was sitmulated by a rather broad range (5–25 mM) of Mg²⁺ and Mn²⁺. NaCl and substrate concentrations also influenced the activity. Pyrophosphate, ATP, inorganic phosphate and NaF inhibited the activity in a dose-dependent manner. Trifluoperazine, chlorpromazine, dibucaine and Triton X-100 (above 0.05%, w/v) specifically inhibited the Ca²⁺-dependent protein kinase in plasma membranes. Repetitive phosphorylation of intrinsic and extrinsic proteins by the membrane-bound enzyme activities clearly showed an important co-ordination of them at the step of protein phosphorylation. These findings suggest that these enzyme activities in plasma membranes may contribute to regulation of thyroid function in response to external stimuli.     

Pyrroline-5-carboxylic acid reductase from soybean leaves

Pyrroline-5-carboxylic acid reductase was purified 40-fold from soybean leaves (Glycine max L. var Corsoy). The enzyme was fairly unstable, had a broad pH optimum, and was inactivated by heat and acid; NADH and NADPH both served as cofactors. It had a higher activity with NADH (about 4 ×) compared to NADPH, but a lower K_m for NADPH. NADP⁺ inhibited both the NADH- and NADPH-dependent activity. Sulfhydryl group blocking agents reduced the activity as did the carbonyl blocking agent, NH₂OH. Thiazolidine-4-carboxylic acid and phosphate inhibited the enzyme and proline inhibited only at high concentrations. ATP, GTP, and CTP were all effective inhibitors of both the NADH- and NADPH-dependent activity. Phosphorylated nucleotide inhibition was reversed by Mg²⁺ ions.     

Catalytic properties of three lactate dehydrogenases from potato tubers (Solanum tuberosum)

Two l-lactate dehydrogenase isoenzymes and one dl-lactate dehydrogenase could be separated from potato tubers by polyacrylamide-gel electrophoresis. The enzymes are specific for lactate, while β-hydroxybutyric acid, glycolic acid, and glyoxylic acid are not oxidized. Their pH optima are pH 6.9 for the oxidation and 8.0 for the reduction reaction.The K_m values for l-lactate for the two isoenzymes are 2.00 × 10^?2 and 1.82 × 10^?2, m. In the reverse reaction the affinities for pyruvate are 3.24 × 10^?4 and 3.34 × 10^?4, m. Both enzymes have similar affinities for NAD and NADH (3.00 × 10^?4; 4.00 × 10^?4, and 8.35 × 10^?4; 5.25 × 10^?4, m).The dl-lactate oxidoreductase may transfer electrons either to NAD or N-methyl-phenazinemethosulfate. The K_m values of this enzyme for l-lactate are 4.5 × 10^?2, m and for d-lactate 3.34 × 10^?2, m. Its affinity for pyruvate is 4.75 × 10^?4, m. The enzyme is inhibited by excess NAD (K_m = 1.54 × 10^?4, M) and has an affinity toward NADH (K_m = 5.00 × 10^?3, M) which is about one tenth of that of the two isoenzymes of l-lactate dehydrogenase.     

Corticotropin stimulates cyclic nucleotide independent protein kinase activity of intact adrenocortical cells

Intact adrenocortical cells possess cyclic nucleotide-independent protein kinase activity which is capable of phosphorylating endogenous proteins and casein when incubated in the presence of [γ-³²P]ATP. The cyclic nucleotide-independent enzyme was dependent on cell number and temperature and had an apparent K_m for ATP of 6.5 × 10^?5 M and a V_max of 12.5 pmol/3 min/2 × 10⁵ cells at 37°C. Phosphorylation of endogenous proteins by this kinase was increased by treatment of intact cells with corticotropin (2.2 nM) for 24 h. In control cells, two endogenous proteins of apparent molecular weights of 39,000 and 76,000 were phosphorylated. In corticotropin-treated cells, another protein of apparent molecular weight of 87,000 was also phosphorylated. Thus, this protein kinase activity, which appears to be located on the plasma membrane, may be involved in mediating longer term actions of corticotropin on the adrenal cortex.     

Plant adenosine kinase: purification and some properties of the enzyme from Lupinus luteus seeds.

Adenosine kinase (ATP:adenosine 5′-phosphotransferase, EC 2.7.1.20) from Lupinus luteus seeds has been obtained with good yield in almost homogeneous state by ammonium sulfate fractionation, chromatography on aminohexyl-Sepharose, and gel filtration. Active enzyme is a single polypeptide chain with a molecular weight of about 38,000 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel nitration. Estimated molecular activity is 156. The enzyme exhibits a strict requirement for divalent metal ions. Among several ions tested the following appeared to be active as cofactors: Co²⁺ ? Mn²⁺ > Mg²⁺ = Ca²⁺ ? Ni²⁺ > Ba²⁺. The optimal metal ion concentrations were as follows: Mn²⁺, 0.5 mm, Mg²⁺ and Ca²⁺, 1 mm, Co²⁺, 1.5 mm. The adenosine kinase shows optimum activity at pH 7.0–7.5. K_m values for adenosine and ATP are 1.5 × 10^?6 and 3 × 10^?4m, respectively. Lupin adenosine kinase is completely inhibited by antisulfhydryl reagents. ATP is the main phosphate donor and among other nucleoside triphosphates ITP, dATP, GTP, and XTP can substitute it but less effectively. Among the ribo- and deoxyribonucleosides occurring in nucleic acids adenosine is phosphorylated effectively and 2′-deoxyadenosine at a lower rate. Of other adenosine analogs tested all adenine d-nucleosides and purine derivative ribosides, besides those with a hydroxyl group at C-6, were found to be substrates for lupin adenosine kinase. Pyrimidine ribo- and deoxyribonucleosides were not phosphorylated.     

Cholinephosphotransferase activity during development of the sea urchin, Arbacia punctulata

Cholinephosphotransferase activity was examined during early development of Arbacia punctulata embryos. CMP was rapidly incorporated by enzyme preparation from Arbacia embryos into a compound identified as CDP-choline. Activity was dependent upon the presence of Mg²⁺, Mn²⁺, or Co²⁺, and was maximal in phosphate buffer, pH 7.0, containing 3 × 10^?2M MgCl₂ and 2 × 10^?5M CMP. Addition of ATP or egg lecithin had no effect on enzyme activity. The activity was localized in the 1000 g and 10,000 g pellets, with little or no activity in the microsomal fraction.Upon fertilization, cholinephosphotransferase activity decreased rapidly, detectable changes in activity being observed within 2 min after fertilization. After reaching a minimum activity 2 hr after fertilization, the enzyme activity increased up to the gastrula stage, then decreased once more. Possible interference by competing enzymes was examined and found to be negligible.     

Physicochemical and Enzymatic Properties of ATP: Nucleotide Pyrophosphotransferase

The molecular weight determined by the sedimentation equilibrium and SDS Polyacrylamide gel electrophoresis was 29,000 and 28,000, respectively. Isoelectric point of the enzyme was determined as pH 7.7. This enzyme contained large amounts of alanine, aspartic acid, glutamic acid and serine, and no cysteine residue was found. The enzyme was inhibited by SDS, KMnO₄, EDTA and tetracycline. GTP and GDP were the most active as pyrophosphate acceptor to the enzyme. The apparent Km for ATP was 2.2×10^?4 m and that for GTP was 2.1×10^?4m in the reaction of ATP+GTP→AMP+pppGpp. On the other hand, in the reaction of 2ATP→AMP+pppApp, the apparent Km for donor and acceptor ATP was 1.7×10^?3m. Effects of pH and metal ions on the enzymatic synthesis of pppGpp were also studied.     

Immobilization of pig muscle 3-phosphoglycerate kinase

3-Phosphoglycerate kinase (ATP:3-phospho-d-glycerate 1-phosphotransferase, EC 2.7.2.3) has been covalently immobilized on a polyacrylamide-type support containing carboxylic groups activated by water-soluble carbodiimide. The activity was 88 units g^?1 xerogel. The activity versus pH profile showed a sharper maximum at pH 6.5 in the case of the immobilized enzyme. The immobilized enzyme had a broad apparent optimum temperature range between 40 and 50°C. The apparent K_m values of the immobilized 3-phosphoglycerate kinase were lower for both 3-phosphoglycerate and ATP than those of the soluble enzyme. In the case of the immobilized enzyme stabilities were enhanced.     

Phosphatidohydrolase and base-exchange activity of commercial phospholipase D

Base-exchange activity was contrasted to the usual phosphatidohydrolase activity of commercial phospholipase D preparation from cabbage. The former activity was assayed by measuring the incorporation of labeled ethanolamine and choline into phospholipids. The latter activity was assayed by measuring the formation of phosphatidic acid with radioactive phosphatidylcholine microdispersion as substrate. The pH optimum for the base-exchange activity was about 9.0, whereas the phosphatidohydrolase activity had a pH optimum around 5.6. The incorporation of ethanolamine and choline into phospholipid was dependent upon the amount of acceptor asolectin microdispersion present. The optimum concentration of Ca²⁺ in the base-exchange reaction was about 4 mm, whereas the optimum concentration for the phosphatidohydrolase activity was greater than 28 mm. The incorporation of ethanolamine into phospholipid was decreased 50% by heating the enzyme preparation at 50°C for about 10 min, whereas the choline incorporation decreased approximately 20% and the phosphatidohydrolase activity decreased by about 10% under these conditions.Hemicholinium-3 was found to be a noncompetitive inhibitor for the incorporation of both ethanolamine and choline into phospholipid with respective K_i, values of 1.25 × 10^?3 and 2.50 × 10^?3m. The K_m values for ethanolamine and choline in the base-exchange reaction were 1.25 × 10^?3 and 2.50 × 10^?3m, respectively. The apparent K_m for phosphatidylcholine for the phosphatidohydrolase activity was about 1.5 × 10^?3m, and there was no inhibition by hemicholinium-3.     

High tyrosine-specific protein kinase activity in normal human peripheral blood lymphocytes

Tyrosine-specific protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) activity was measured in normal human nonadherent peripheral blood lymphocytes using synthetic peptide substrates having sequence homologies with either pp60^src or c-myc. A high level of tyrosine-specific protein kinase activity was found associated with the cell particulate fraction (100 000 × g pellet). High-pressure liquid chromatography and phosphoamino acid analysis of the synthetic peptide substrates substantiated the phosphorylation of tyrosine residues by the particulate fraction enzyme. The human enzyme was also capable of phosphorylating a synthetic random polymer of 80% glutamic acid and 20% tyrosine. Enzyme activity was half-maximal with 22 μM Mg·ATP and had apparent K_m values for the synthetic peptides from 1.9 to 7.1 mM. The enzyme preferred Mg²⁺ to Mn²⁺ for optimal activity and was stimulated 2–5-fold by low levels (0.05%) of some ionic as well as non-ionic detergents including deoxycholate, Nonidet P-40 and Triton X-100. The enzyme activity was not stimulated by N⁶;O^2′-dibutyryl cyclic AMP (100 μM), N⁶;O^2′-dibutyryl cyclic GMP (100 μM), Ca²⁺ (200 μM), insulin (1 μg/ml) or homogeneous human T-cell growth factor (3 μg/ml) under the conditions used. Alkaline-resistant phosphorylation of particulate proteins in vitro revealed protein bands with M_r 59 000 and 54 000 suggesting that there are endogenous substrates for the human lymphocyte tyrosine protein kinase.     

Affinity labelling of tryptophanyl-transfer RNA synthetase

A double affinity-labelling approach has been developed in order to convert an oligomeric enzyme with multiple active centres into a single-site enzyme.Tryptophanyl-transfer RNA synthetase (EC 6.1.1.2) from beef pancreas is a symmetric dimer, α₂ An ATP analogue, γ-(p-azidoanilide)-ATP does not serve as a substrate for enzymatic aminoacylation of tRNA^Trp but acts as an effective competitive inhibitor in the absence of photochemical reaction, with K₁ = 1 × 10^?3m (K_mfor ATP = 2 × 10^?4m). The covalent photoaddition of azido-ATP³ results in complete loss of enzymatic activity in both the ATP-[³²P]pyrophosphate exchange reaction and tRNA aminoacylation. ATP completely protects the enzyme against inactivation. However, covalent binding of azido-ATP is also observed outside the active centres. The difference between covalent binding of the azido-ATP in the absence and presence of ATP corresponds to 2 moles of the ATP analogue per mole of the enzyme.Two binding sites for tRNA^Trp have been found from complex formation at pH 5.8 in the presence of Mg²⁺. The two tRNA molecules bind, with K_dis = 3.6 × 10^?8m and K_dis = 0.9 × 10^?6m, respectively, pointing to a strong negative co-operativity between the binding sites for tRNA.N-chlorambucilyl-tryptophanyl-tRNA^Trp and TRSase form a complex with K_dis = 5.5 × 10^?8m at pH 5.8 in the presence of 10 mm-Mg²⁺. This value is similar to the value of K_dis for tryptophanyl-tRNA of 4.8 × 10^?8m. Under the same conditions a 1:1 complex (in mol) is formed between the enzyme and Trp-tRNA or N-chlorambucilyl-Trp-tRNA. On incubation, a covalent bond is formed between N-chlorambucilyl-Trp-tRNA and TRSase; 1 mole of affinity reagent alkylates 1 mole of enzyme independently of the concentration of the modifier. The alkylation reaction is completely inhibited by the presence of tRNA^Trp whereas the tRNA devoid of tRNA^Trp does not affect the rate of alkylation. In the presence of either ATP or tryptophan, or a mixture of the two, the alkylation reaction is inhibited even though these ligands have no effect on the complex formation between TRSase and the tRNA analogue. Photoaddition of the azido-ATP completely prevents the reaction of the enzyme with the tRNA analogue, although the non-covalent complex formation is not affected.Exhaustive alkylation of TRSase partially inhibits the reaction of ATP [³²P]pyrophosphate exchange and completely blocks the aminoacylation of tRNA^Trp. Cleavage of the tRNA which is covalently bound to TRSase restores both the ATP-[³²P]pyrophosphate exchange and aminoacylation activity.The TRSase which is covalently-bound to R-Trp-tRNA is able to incorporate only one ATP molecule per dimeric enzyme into the active centre. This doubly modified enzyme is completely enzymatically inactive. Removal of the tRNA residue from the doubly modified enzyme results in the formation of the derivative with one blocked ATP site. Therefore, a “single-site” TRSase may be generated either by alkylation of the enzyme with Cl-R-Trp-tRNA or after the removal of covalently bound tRNA from the doubly labelled protein.Tryptophanyl-tRNA synthetase containing blocked ATP and/or tRNA binding site(s) seems to bo a useful tool for investigation of negative co-operativity and may help in the elucidation of the structure function relationships between the active centres.