Functional arginine residues and carboxyl groups in the adenosine triphosphatase of the thermophilic bacterium PS-3

Treatment of purified ATPase of the thermophilic bacterium PS-3 with the arginine reagent phenylglyoxal or with Woodward's reagent K, gave complete inactivation of the enzyme. The inactivation rates followed apparent first-order kinetics. The apparent order of reaction with respect to inhibitor concentrations gave values near to 1 with both reagents, suggesting that inactivation was a consequence of modifying one arginine or carboxyl group per active site. ADP and ATP strongly protected the thermophilic ATPase against both reagents. GDP and IDP protected less, whilst CTP did not protect. Experiments in which the incorporation of [14C]phenylglyoxal into the enzyme was measured show that extrapolation of incorporation to 100% inactivation of the enzyme gives 8-9 mol [14C]phenylglyoxal per mol ATPase, whilst ADP or ATP prevent modification of about one arginine per mol.     

Chemical modification of the F0 part of the ATP synthase (F1F0) from Escherichia coli. Effects on proton conduction and F1 binding

The purified F0 part of the ATP synthase complex from Escherichia coli was incorporated into liposomes and chemically modified by various reagents. The modified F0-liposomes were assayed for H+ uptake and, after reconstitution with F1, for total and dicyclohexylcarbodiimide-sensitive ATPase activity. The water-soluble carbodiimide, 1-ethyl-3-(-3-dimethylaminopropyl)carbodiimide methiodide, (1.2 mM), inhibited H+ uptake to a great extent. Binding of F1 was almost unaffected, but the hydrolysis of ATP was uncoupled from H+ transport. This is reflected by the inhibition of dicyclohexylcarbodiimide-sensitive ATPase activity. Woodward's reagent K, N-ethyl-5-phenylisoxazolium-3'-sulfonate, inhibited both H+ uptake and total ATPase activity. Modification of arginine residues by phenylglyoxal (20 mM) was followed by inhibition of the F1 binding activity by 80% of the control. H+ translocation was reduced to 70%. Diethylpyrocarbonate (3 mM) exhibited a strong inhibiting effect on H+ uptake but not on F1 binding. Modification of tyrosine (by tetranitromethane) as well as lysine residues (by succinic anhydride) did not affect F0 functions. From the data presented we conclude that carboxyl-groups, different from the dicyclohexylcarbodiimide-binding site, are involved in H+ translocation through F0 and, in part, in the functional binding of F1. Furthermore, for the latter function, also arginine residues seem to be important. The role of histidine residues remains unclear at present.     

Involvement of arginine residues in catalysis by rat brain hexokinase

Inactivation of rat brain hexokinase (ATP:d-hexose 6-phosphotransferase, EC 2.7.1.1) by the arginine-specific reagent, phenylglyoxal, has been studied. Inactivation did not follow pseudo-first-order kinetics, suggesting the involvement of two or more arginine residues in catalytic function. Using [¹⁴C]phenylglyoxal, it was found that 5 of the 55 arginines per molecule of hexokinase react with this reagent, with an accompanying loss of over 90% of the catalytic activity. Virtually all of the activity loss occurs during derivatization of four relatively slower reacting arginines, with essentially no activity loss during derivatization of one rapidly reacting arginine. Inactivation by phenylglyoxal was not due to reaction with critical sulfhydryl groups in brain hexokinase since reactivity of the enzyme with the sulfhydryl reagent, 5,5′-dithiobis(2-nitrobenzoic acid) was not affected by prior treatment with phenylglyoxal. Comparison of amino acid composition, before and after reaction with phenylglyoxal, indicated that only the arginine content had been affected by phenylglyoxal treatment. The decrease in arginine content, measured by amino acid analysis, and the incorporation of phenylglyoxal, measured with [¹⁴C]phenylglyoxal, was consistent with the phenylglyoxal:arginine stoichiometry of 2:1 originally reported by K. Takahashi (1968, J. Biol. Chem.243, 6171–6179). Several ligands were tested and found to provide varying degrees of protection of hexokinase activity against phenylglyoxal. ATP and ADP alone provided only slight protection, but were highly effective in the presence of N-acetylglucosamine which itself gave only moderate protection. Glucose 6-phosphate and 1,5-anhydroglucitol 6-phosphate, both good inhibitors of brain hexokinase, were very effective while poorly inhibitory hexose 6-phosphates were not. Glucose was very effective, with protection afforded by other hexoses being correlated with their ability to serve as substrates (i.e., poor substrates also provided little protection against phenylglyoxal). The effectiveness of hexose 6-phosphates and hexoses in protecting the enzyme against inactivation by phenylglyoxal was related to their ability to induce conformational change in the enzyme. None of the ligands tested appreciably affected the reactivity of the rapidly reacting arginine residue. There was no correlation between the inhibition observed in the presence of various ligands and the number of arginines reacted with phenylglyoxal. The results were interpreted as indicating the involvement of two to four arginine residues in the catalytic function of brain hexokinase, possibly in the binding of anionic ligands such as ATP, ADP, or glucose 6-phosphate.     

Structure-function investigations of the membrane (Na+ + Mg2+)-ATPase from Acholeplasma laidlawii B: studies of reactive amino acid residues using group-specific reagents

The purified, lipid-reconstituted (Na+ + Mg2+)-ATPase from Acholeplasma laidlawii B was treated with a variety of reagents which specifically modify various amino acid residues on the enzyme. In all cases reaction of this enzyme with any of the reagents tested results in at least a partial inactivation of its activity. The modification of one reactive lysine by dinitrofluorobenzene, of one reactive arginine by phenylglyoxal, or of two tyrosine residues by 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole or fluorosulfonylbenzoyl adenosine results in a complete inactivation of the enzyme. Partial inactivation of enzymatic activity with N-ethylmaleimide, p-chloromercuribenzene sulfonic acid, dicyclohexylcarbodiimide, and Woodward's reagent K suggests an indirect involvement of sulfhydryl and carboxylic acid groups in the maintenance of enzymatic activity, although inhibition by these reagents may also be the result of nonspecific effects such as subunit crosslinking. These studies also show that all of the subunits of the ATPase can be labeled by aqueous-phase reagents directed at amino groups and phenolic groups, and provide evidence for a specific affinity labeling of the alpha subunit of the enzyme by a nucleotide analog directed at phenolic and/or sulfhydryl groups.     

Arginine-specific modification of rabbit muscle phosphoglucose isomerase: Differences in the inactivation by phenylglyoxal and butanedione and in the protection by substrate analogs

Rabbit muscle phosphoglucose isomerase was modified with phenylglyoxal or 2,3-butanedione, the reaction with either reagent resulting in loss of enzymatic activity in a biphasic mode. At slightly alkaline pH butanedione was found to be approximately six times as effective as phenylglyoxal. The inactivation process could not be significantly reversed by removal of the modifier. Competitive inhibitors of the enzyme protected partially against loss of enzyme activity by either modification. The only kind of amino acid residue affected was arginine. However, more than one arginine residue per enzyme subunit was found to be susceptible to modification by the dicarbonyl reagents. From protection experiments it was concluded (i) that both modifiers react specifically with an arginine in the phosphoglucose isomerase active site and nonspecifically with one or more arginine residues elsewhere in the enzyme molecule, (ii) that modification at either loci causes loss of catalytic activity, and (iii) that butanedione has a higher preference for active site arginine than for arginine residues outside of the catalytic center whereas the opposite is true for phenylglyoxal.     

Reaction of phenylglyoxal with chicken gizzard myosin

Modification of chicken gizzard myosin with phenyl[2-14C]-glyoxal inhibited the K+-ATPase (ATP phosphohydrolase, EC 3.6.1.32) activity as a function of time. During the 2.5 and 15 min interval 3.2 mol of the reagent were incorporated per 4.7 X 10(5) g protein and the K+-ATPase activity was 50% inhibited. Phenylglyoxal reacted with arginine residues of gizzard myosin in a mol ratio of two to one, phenylglyoxal to arginine as determined spectrophotometrically. The modification was limited to the subfragment 1 heavy chain and rod-like regions and none of the light chains were lost. The inhibition of the ATPase activity occurred when the subfragment 1 region was modified predominantly. The same results were obtained when the myosin was phosphorylated and then incubated with phenylglyoxal. Substrate MgATP2- or MgADP enhanced the inactivation of gizzard myosin; there was an increase in the incorporation of the reagent and a change in the distribution into the heavy chains. Approx. 0.5 mol of the nucleotide was bound to 4.7 X 10(5) g of phenylglyoxal myosin. Conformational changes, induced by these modifications, were responsible for the inhibition of enzymic activity. Arginine residues of gizzard myosin are necessary for the maintenance of the ATPase activity of this contractile protein.     

Evidence for an essential arginine residue at the active site of ATP citrate lyase from rat liver.

Rat liver ATP citrate lyase was inactivated by 2, 3-butanedione and phenylglyoxal. Phenylglyoxal caused the most rapid and complete inactivation of enzyme activity in 4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid buffer, pH 8. Inactivation by both butanedione and phenylglyoxal was concentration-dependent and followed pseudo- first-order kinetics. Phenylglyoxal also decreased autophosphorylation (catalytic phosphate) of ATP citrate lyase. Inactivation by phenylglyoxal and butanedione was due to the modification of enzyme arginine residues: the modified enzyme failed to bind to CoA-agarose. The V declined as a function of inactivation, but the Km values were unaltered. The substrates, CoASH and CoASH plus citrate, protected the enzyme significantly against inactivation, but ATP provided little protection. Inactivation with excess reagent modified about eight arginine residues per monomer of enzyme. Citrate, CoASH and ATP protected two to three arginine residues from modification by phenylglyoxal. Analysis of the data by statistical methods suggested that the inactivation was due to modification of one essential arginine residue per monomer of lyase, which was modified 1.5 times more rapidly than were the other arginine residues. Our results suggest that this essential arginine residue is at the CoASH binding site.     

A reactive arginine in adenylate kinase.

Adenylate kinase (ATP:AMP phosphotransferase, EV 2.7.4.3) from pig heart is inactivated by the specific arginyl reagent phenylglyoxal. During inactivation two molecules of phenyglyoxal are incorporated into the protein indicating the modification of one of the 11 arginine residues. The modification of other amino acids is ruled out. Chemical modification of this essential residue is prevented by high concentrations of the substrates AMP, ADP and MgATP2-. The protection of the substrates is explained by the formation of a ternary abortive enzyme-substrate complex ESS. The dissociation constants KD = [ES] - [S]/[ESS] are determined from the kinetic data of inactivation and protection.     

Functional carboxylic group in the active center of transketolase

Baker's yeast transketolase is rapidly inactivated in the presence of carboxylic group modifiers, i.e., 1-ethyl-3(3'-dimethylaminopropyl)-carbodiimide or Woodward's reagent K. This inactivation is due to modification of the carboxylic group in the enzyme active center. The essential groups localized in the two active centers of transketolase differ in the rate of modification; accordingly, the inactivation kinetics appears as biphasic. A complete loss of the enzyme activity occurs as a result of modification of one carboxylic group per enzyme active center. The pKa value of modifiable groups is equal to about 6.5. This modification decreases by two orders of magnitude the affinity of the substrate for the active center. The carboxylic groups are not directly involved in the interaction with the substrates; their modification does not significantly affect the coenzyme binding. It is supposed that these groups are responsible for the deprotonation of the second carbon in the thiamine pyrophosphate thiazolium ring.     

Essential arginine residues in the active sites of propionyl CoA carboxylase and beta-methylcrotonyl CoA carboxylase

At least one arginine residue is essential for substrate binding in or near the active sites of propionyl CoA carboxylase (PCC) and beta-methylcrotonyl CoA carboxylase (beta MCC) in cultured human fibroblasts. This conclusion is based on studies of enzyme inhibition by phenylglyoxal, a reagent which specifically modifies arginine residues. Human fibroblast PCC both in extracts and in a 20-fold purified preparation was nearly completely protected from phenylglyoxal inhibition following incubation with propionyl CoA or ATP. It appears that a phosphate group from either ATP or the CoA moiety of propionyl CoA reacts with the essential arginine residue(s). beta MCC which was similarly inhibited by phenylglyoxal was protected by beta-methylcrotonyl CoA and ATP. Thus phenylglyoxal may be used to label specific arginine residues within the active sites of previously sequenced carboxylases.     

Effects of protein-modifying reagents on an isoenzyme of potato apyrase

Treatment of an isoenzyme of potato apyrase of high adenosine triphosphatase/adenosine diphosphatase (ATPase/ADPase) ratio with iodine, N-acetylimidazole or tetranitromethane inactivates the ATPase activity of this enzyme faster than its ADPase activity. There was protection by substrates with the two last-named substances. This and the appearance of nitrotyrosine suggests the participation of tyrosyl residues in both enzymic activities of potato apyrase. The participation of thiol groups is excluded by the insensitivity of apyrase to p-chloromercuribenzoate. Also, 2-hydroxy-5-nitrobenzyl bromide or carboxymethylation produce the same rate of inactivation of ATPase and ADPase activities. Substrates protect both activities from inactivation. Hydrogen peroxide and photo-oxidation inactivate ATPase activity faster than ADPase activity. There is no protection by substrates. Analysis of pH effects on V_max. and K_m suggest different pK values for the amino acid residues at the ATP and ADP sites.     

Glutamate 59 is critical for transport function of the amino acid cotransporter KAAT1

KAAT1 is a neutral amino acid transporter activated by K⁺ or by Na⁺ (9). The protein shows significant homology with members of the Na⁺/Cl^–-dependent neurotransmitter transporter super family. E59G KAAT1, expressed in Xenopus oocytes, exhibited a reduced leucine uptake [20–30% of wild-type (WT)], and kinetic analysis indicated that the loss of activity was due to reduction of V_max and apparent affinity for substrates. Electrophysiological analysis revealed that E59G KAAT1 has presteady-state and uncoupled currents larger than WT but no leucine-induced currents. Site-directed mutagenesis analysis showed the requirement of a negative charge in position 59 of KAAT1. The analysis of permeant and impermeant methanethiosulfonate reagent effects confirmed the intracellular localization of glutamate 59. Because the 2-aminoethyl methanethiosulfonate hydrobromid inhibition was not prevented by the presence of Na⁺ or leucine, we concluded that E59 is not directly involved in the binding of substrates. N-ethylmaleimide inhibition was qualitatively and quantitatively different in the two transporters, WT and E59G KAAT1, having the same cysteine residues. This indicates an altered accessibility of native cysteine residues due to a modified spatial organization of E59G KAAT1. The arginine modifier phenylglyoxal effect supports this hypothesis: not only cysteine but also arginine residues become more accessible to the modifying reagents in the mutant E59G. In conclusion, the results presented indicate that glutamate 59 plays a critical role in the three-dimensional organization of KAAT1. amino acid transport; structure/function; amino acid modifiers; Manduca sexta     

Chemical modification studies on arginine kinase: essential cysteine and arginine residues at the active site

Chemical modification was used to elucidate the essential amino acids in the catalytic activity of arginine kinase (AK) from Migratoria manilensis. Among six cysteine (Cys) residues only one Cys residue was determined to be essential in the active site by Tsou's method. Furthermore, the AK modified by DTNB can be fully reactivated by dithiothreitol (DTT) in a monophasic kinetic course. At the same time, this reactivation can be slowed down in the presence of ATP, suggesting that the essential Cys is located near the ATP binding site. The ionizing groups at the AK active site were studied and the standard dissociation enthalpy (ΔH°) was 12.38 kcal/mol, showing that the dissociation group may be the guanidino of arginine (Arg). Using the specific chemical modifier phenylglyoxal (PG) demonstrated that only one Arg, located near the ATP binding site, is essential for the activity of AK.     

Identification of an essential arginine residue in the beta subunit of the chloroplast ATPase

The ATPase activity of soluble chloroplast coupling factor (CF1) was irreversibly inactivated by phenylglyoxal, an arginine reagent. Under the conditions of inactivation, 2.48 mol of [14C]phenylglyoxal were incorporated per 400,000 g of enzyme when the ATPase was inactivated 50% by the reagent. Isolation of the component polypeptide subunits of the [14C]phenylglyoxal-modified enzyme revealed that the distribution of moles of labeled reagent/mol of subunit was the following: alpha, 0.37; beta, 0.40; gamma, 0.08; delta, none; epsilon, 0.03. CNBr treatment of the isolated alpha and beta subunits and fractionation of the peptides by gel electrophoresis revealed that the radioactivity bound to the alpha subunit was nonspecifically associated with several peptides, while a single peptide derived from the beta subunit contained the majority of the radioactivity associated with this subunit. After treating the isolated beta subunit with trypsin and Staphylococcus aureus protease, a major radioactive peptide was isolated with a sequence Arg-Ile-Thr-Ser-Ile-Lys. This sequence, when compared with the primary structure of the CF1 beta subunit as translated from the gene (Zurawski, G., Bottomley, W., and Whitfeld, P. R. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 6260-6264) indicated that the arginine marked with the asterisk, the predominant residue modified by phenylglyoxal when the ATPase activity of CF1 is inactivated by the reagent, is Arg 312.     

Evidence for an essential arginine residue at the active site of Escherichia coli acetate kinase

Escherichia coli acetate kinase (ATP: acetate phosphotransferase, EC 2.7.2.1.) was inactivated in the presence of either 2,3-butanedione in borate buffer or phenylglyoxal in triethanolamine buffer. When incubated with 9.4 mM phenylglyoxal or 5.1 mM butanedione, the enzyme lost its activity with an apparent rate constant of inactivation of 0.079 min-1, respectively. The loss of enzymatic activity was concomitant with the loss of an arginine residue per active site. Phosphorylated substrates of acetate kinase, ATP, ADP and acetylphosphate as well as AMP markedly decreased the rate of inactivation by both phenylglyoxal and butanedione. Acetate neither provided any protection nor affected the protection rendered by the adenine nucleotides. However, it interfered with the protection afforded by acetylphosphate. These data suggest that an arginine residue is located at the active site of acetate kinase and is essential for its catalytic activity, probably as a binding site for the negatively charged phosphate group of the substrates.     

Involvement of arginine residues in the allosteric activation and inhibition ofSynechocystis PCC 6803 ADPglucose pyrophosphorylase

ADPglucose pyrophosphorylase (EC 2.7.7.27) from the cyanobacteriumSynechocystis PCC 6803 was desensitized to the effects of allosteric ligands by treatment with the arginine reagent, phenylglyoxal. Enzyme modification by phenylglyoxal resulted in inactivation when the enzyme was assayed under 3P-glycerate-activated conditions. There was little loss of the catalytic activity assayed in the absence of activator. Pi, 3P-glycerate, and pyridoxal-P were able to protect the enzyme from inactivation, whereas substrates gave minimal protection. The protective effect exhibited by Pi and 3P-glycerate was dependent on effector concentration. MgCl₂ enhanced the protection afforded by 3P-glycerate. The enzyme partially modified by phenylglyoxal was more resistant to 3P-glycerate activation and Pi inhibition than the unmodified form.V _max at saturating 3P-glycerate concentrations and the apparent affinity of the enzyme toward Pi were decreased upon phenylglyoxal modification. Incorporation of labeled phenylglyoxal into the enzyme was proportional to the loss of activity. Pi and 3P-glycerate nearly completely prevented incorporation of the reagent to the protein. Results suggest that one arginine residue per mol of enzyme subunit is involved in the binding of allosteric effector in the cyanobacterial ADPglucose pyrophosphorylase.     

Molecular characteristics of small intestinal and renal brush border thiamin transporters in rats.

The molecular characteristics of thiamin (T) transport were studied in the small intestinal and renal brush border membrane vesicles of rats, using [(3)H]T at high specific activity. The effects of various chemical modifiers (amino acid blockers) on T uptake were examined and their specificity assessed. Treatment with the carboxylic specific blockers 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide metho-p-toluene sulfonate, (1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide hydrochloride and N-ethyl-5-phenylisoaxolium-3'-sulfonate (Woodward's Reagent K) and with the sulfhydryl specific blocker p-chloromercuribenzene sulfonate inhibited T transport in both types of vesicles. Phenylglyoxal, but not ninhydrin, both reagents for arginine residues, and diethylpyrocarbonate, a reagent for histidine residues, specifically decreased T transport only in renal and small intestinal vesicles respectively. Similarly 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole reacted, but not N-acetylimidazole, both of which are reagents for tyrosine residues. However, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole inhibition was aspecific. Acetylsalicylic acid, a reagent for lysine and serine residues, decreased T transport, but the lysine effect was aspecific. Acetylsalicylic acid serine blockage also eliminated T/H(+) exchange in small intestinal vesicles. Taken together, these results suggest that for T transport carboxylic and sulfhydryl groups and serine residues are essential in both renal and small intestinal brush border membrane vesicles. In addition, arginine and histidine residues are also essential respectively for renal and small intestinal transporters. Serine was essential for the T/H(+) antiport mechanism.     

Involvement of arginine residues in the allosteric activation and inhibition ofSynechocystis PCC 6803 ADPglucose pyrophosphorylase

ADPglucose pyrophosphorylase (EC 2.7.7.27) from the cyanobacteriumSynechocystis PCC 6803 was desensitized to the effects of allosteric ligands by treatment with the arginine reagent, phenylglyoxal. Enzyme modification by phenylglyoxal resulted in inactivation when the enzyme was assayed under 3P-glycerate-activated conditions. There was little loss of the catalytic activity assayed in the absence of activator. Pi, 3P-glycerate, and pyridoxal-P were able to protect the enzyme from inactivation, whereas substrates gave minimal protection. The protective effect exhibited by Pi and 3P-glycerate was dependent on effector concentration. MgCl₂ enhanced the protection afforded by 3P-glycerate. The enzyme partially modified by phenylglyoxal was more resistant to 3P-glycerate activation and Pi inhibition than the unmodified form.V _max at saturating 3P-glycerate concentrations and the apparent affinity of the enzyme toward Pi were decreased upon phenylglyoxal modification. Incorporation of labeled phenylglyoxal into the enzyme was proportional to the loss of activity. Pi and 3P-glycerate nearly completely prevented incorporation of the reagent to the protein. Results suggest that one arginine residue per mol of enzyme subunit is involved in the binding of allosteric effector in the cyanobacterial ADPglucose pyrophosphorylase.     

Two types of essential carboxyl groups in Rhodospirillum rubrum proton ATPase

Two different types of essential carboxyl groups were detected in the extrinsic component of the proton ATPase of Rhodospirillum rubrum. Chemical modification of R. rubrum chromatophores or its solubilized ATPase by Woodward's reagent K resulted in inactivation of photophosphorylating and ATPase activities. The apparent order of reaction was nearly 1 with respect to reagent concentration and similar K1 were obtained for the soluble and membrane-bound ATPases suggesting that inactivation was associated with modification of one essential carboxyl group located in the soluble component of the proton ATPase. Inactivation was prevented by adenine nucleotides but not by divalent cations. Dicyclohexylcarbodiimide completely inhibited the solubilized ATPase with a K1 of 5.2 mM and a K2 of 0.81 min-1. Mg2+ afforded nearly complete protection with a Kd of 2.8 mM. Two moles of [14C]dicyclohexylcarbodiimide were incorporated per mole of enzyme for complete inactivation but in the presence of 30 mM MgCl2 only one mole was incorporated and there was no inhibition. The labeling was recovered mostly from the beta subunit. The incorporation of the labeled reagent into the ATPase was not prevented by previous modification with Woodward's reagent K. It is concluded that both reagents modified two different essential carboxyl groups in the soluble ATPase from R. rubrum.     

A comparative study of essential arginine residues in Gramicidin S synthetase 2 and isoleucyl tRNA synthetase

In gramicidin S synthetase 2 (GS 2) from Bacillus brevis, L-proline, L-valine, L-ornithine, and L-leucine activations to aminoacyl adenylates are progressively inhibited by phenylglyoxal. The inactivation of GS 2 obeys pseudo-first-order kinetics. ATP completely prevents inactivation of GS 2 by phenylglyoxal, whereas amino acids only partially prevent it. In the presence of ATP, four arginine residues per mol of GS 2 are protected from modification by phenylglyoxal as determined by amino acid analysis and the incorporation of [7-14C]phenylgloxal into the enzyme protein, indicating that a single arginine residue is necessary for each amino acid activation. In isoleucyl tRNA synthetase from Escherichia coli, phenylglyoxal inhibits activation of L-isoleucine to isoleucyl adenylate. ATP completely prevents inactivation, although isoleucine only partially prevents it. One arginine residue of isoleucyl tRNA synthetase is protected by ATP from modification by phenylglyoxal, suggesting that a single arginine residue is essential for isoleucine activation. These results support the involvement of arginine residues in ATP binding with GS 2 or isoleucyl tRNA synthetase, and thus indicate that arginine residues of amino acid activating enzymes are essential for the formation of aminoacyl adenylates in both nonribosomal and ribosomal peptide biosynthesis.