31P-NMR saturation transfer study of the in vivo kinetics of arginine kinase in Carcinus crab leg muscle

The kinetics of the reaction catalyzed by arginine kinase have been determined at 9.5 and 23°C for in vivo leg muscle of Carcinus maenas (the common shore crab) using the noninvasive technique of ³¹P-NMR spectroscopy. Concentrations of mobile phosphorus metabolites were the same at both temperatures: 78.7 mM for arginine phosphate, 9.0 mM for adenosine triphosphate (ATP), and 2.6 mM for inorganic phosphate (P_i), as estimated from NMR resonance intensities and literature values for ATP concentration as assayed by traditional biochemical methods. Apparent unidirectional rate constants for formation of ATP from arginine phosphate and ADP were 0.09 s^?1 at 9.5°C and 0.27 s^?1 at 23°C. Pseudo-first-order rate constants for arginine phosphate generation from Arg and ATP were 0.38 and 1.10 s^?1 at 9.5 and 23°C, respectively. In vivo Q₁₀ for the arginine kinase reaction between 9.5 and 23°C was thus 2.2 for both directions. When the kinetic data are analyzed using the Arrhenius equation, activation energies of 126 kJ/mol for ATP formation and 105 kJ/mol for arginine phosphate formation are found. The measured chemical fluxes through arginine kinase in the forward reaction (arginine phosphate hydrolysis) were twice those in the reverse reaction, consistent with either compartmentation of substrates or participation of substrates in alternative metabolic pathways.     

On the role of arginine kinase in insect flight muscle

In the flight muscle of Locusta migratoria L., arginine kinase activity increased 10-fold when 5th instar larvae and adult animals were compared. During the onset of flight, ATP decreased slightly with the amount of phospho-l-arginine remaining constant. Thus, high arginine kinase activity characterizes the adult muscle, giving rise to the speculation that the phospho-l-arginine/l-arginine kinase system does not act only as a buffer system for high-energy phosphate but also as a shuttle mechanism for high-energy phosphate between mitochondria and myofibrils. Judged from electrophoretic mobility, only one isoenzyme exists that is not bound to subcellular structures. Calculations of the diffusive fluxes of ATP, ADP, phosphate, phospho-l-arginine and l-arginine between the sites of ATP-consumption and production, respectively, can be interpreted in such a way, that the low concentration of ADP_free might limit ATP-turnover during flight. Judging from the high arginine kinase activity, the major acceptor for high-energy phosphate at the mitochondria could be l-arginine, while phospho-l-arginine is transphosphorylated to ATP at the myofibrils, thus presumably serving as an energy shuttle.     

Respiration and phosphorylation by mitochondria from the hepatopancreas of the blue crab (Callinectes sapidus)

Mitochondria isolated from the hepatopancreas of the blue crab Callinectes sapidus show high rates of oxidation of pyruvate + proline and of various intermediates of the tricarboxylic acid cycle in a 280- to 380-mOsm sucrose-mannitol medium supplemented with bovine serum albumin. The respiratory control ratio ranged from 6 to 10. Respiration was accompanied by phosphorylation of ADP, with the expected ADP:O ratio for all substrates tested except α-ketoglutarate, indicating that all three energy-conserving sites were functional. Fatty acids were also oxidized, but no oxidation of β-hydroxybutyrate, glycerol 3-phosphate, or NADH was observed. The crab mitochondria showed a relatively low affinity for phosphate, but a normal affinity for ADP. Respiration and phosphorylation gave the normal responses to respiratory chain inhibitors, uncoupling agents, oligomycin, and ionophores. Crab mitochondria have an exceptionally high content of phosphate, exceeding 1000 nmoles per mg protein, but a normal energy charge of the adenylic system. An unusual feature is the presence of considerable arginine kinase activity, which is usually thought to be restricted to muscle and nerve tissue in anthropods. This enzyme allows arginine to act as secondary phosphate acceptor. The arginine kinase is located on the cytosol side of the atractyloside-sensitive barrier and is thus unable to transfer the terminal phosphate group of matrix ATP directly to arginine.     

31P-NMR studies of the freeze-tolerant larvae of the gall fly, Eurosta solidaginis

31P NMR was applied to an examination of the freeze-tolerant larvae of the gall fly, Eurosta solidaginis. Resonances from sugar phosphates, inorganic phosphate, adenylates and arginine phosphate were identified. Two peaks of Pi were identified corresponding to intracellular and extracellular Pi. Anoxia produced an expected decrease in peak intensities of ATP and arginine phosphate while the peak of intracellular Pi was enhanced and shifted to indicate intracellular acidification during anoxia. Spectra of whole larvae were monitored over a temperature range from -30 degrees to +25 degrees C. No abrupt alterations in the spectra were seen at the point of extracellular freezing which occurs at about -8 degrees C but temperature had dramatic effects upon the peak intensities of ATP and arginine phosphate. A reversible increase/decrease in peak intensities, relative to Pi, was observed as temperature was raised/lowered. At 15 degrees and -20 degrees C, the beta peak of ATP was 64% and 2% of the peak intensity of Pi while that of arginine phosphate was 78% and 11%, respectively. This temperature effect was not an artifact of instrumentation (as model solutions containing Pi, ATP and arginine phosphate did not show this effect) or a result of changes in the total amounts of these compounds in the cell with temperature. Rather it is apparent that these molecules become restricted in their rotational movement as temperature is lowered perhaps via binding to subcellular components. Changes in the amounts of freely soluble ATP and arginine phosphate with temperature could have important implications for metabolism and its control. Analysis of the effect of temperature on the chemical shift of Pi was also used to determine pH in the intracellular and extracellular compartments. Temperature change had no effect on extracellular (hemolymph) pH which remained constant at 6.1-6.3. Intracellular pH varied with temperature, however, from pH 6.8 at 15 degrees C to pH 7.3 at -12 degrees C with a change, delta pH/delta 0, of -0.0185 degrees C consistent with alphastat regulation.     

Conformation of triphosphate and lysine-triphosphate-arginine complex

The semi-empirical all-valence electron CNDO/2 MO method has been used to calculate energy states of phosphate and triphosphate made analogous to ATP by adding a methyl group at one end and by making charge transfer complexes with arginine and lysine analogs. The calculations suggest that when ATP is attached to arginine and lysine residues, as is known to occur in actin, ATP is in a low energy state.     

Kinetic properties of arginine phosphokinase from honeybees, Apis mellifera L. (Hymenoptera, Apidae)

The kinetic properties of honeybee arginine phosphokinase (APK), which catalyzes the reaction: Arginine phosphate + ADP + H⁺ ? arginine + ATP, have been studied.In the direction of ATP synthesis, the pH optimum was around pH 7.2 and the activation energy over the range 18–44 °C was about 10,500 cal/mole. The optimum ratio of Mg²⁺:ADP was about 4:1.In the direction of arginine phosphate (AP) synthesis, the enzyme had a pH optimum around pH 8.3. The energy of activation for the reaction over the range 22–39 °C was about 7500 cal/mole. The optimum ratio of Mg²⁺:ATP was about 1:1.The initial velocities of the reactions in the direction of ATP and AP synthesis were measured at varying concentrations of one substrate while the concentration of the other substrate was held constant at several levels. The double reciprocal plots of the data obtained yielded a series of intersecting lines, indicating that the enzyme has a sequential mechanism. Radioisotope exchange experiment showed that arginine phosphokinase did not catalyze ATP ? ADP exchange in the absence of arginine. Product inhibition studies showed that arginine was competitive with AP and noncompetitive with ADP; whereas ATP was competitive with ADP and noncompetitive with arginine. The results from initial velocity, radioisotope exchange, and product inhibition studies suggested that the enzyme has a rapid equilibrium, random mechanism.     

Modification of an essential arginine of carbamate kinase

Carbamate kinase from Streptococcus faecalis is inactivated by butanedione in borate buffer, which implies the presence of an essential arginine at the active site of the enzyme. The inactivation reaction is first order in [butanedione] and a replot of the inactivation rate data infers that one arginine is modified. The enzyme is protected against inactivation by ADP, ATP, the metal-nucleotides and carbamyl phosphate but not by carbamate. Amino acid analyses reveal that one of three arginines is modified by butanedione in the absence of protecting agents, and the binding of ADP to the enzyme prevents modification. Thus, analysis of the data suggest that (i) substrate binding to arginine and (ii) protein conformational changes at the active site are responsible for protection of an essential arginine against modification by butanedione.     

Transport of arginine and aspartic Acid into isolated barley mesophyll vacuoles

The transport of arginine into isolated barley (Hordeum vulgare L.) mesophyll vacuoles was investigated. In the absence of ATP, arginine uptake was saturable with a K_m of 0.3 to 0.4 millimolar. Positively charged amino acids inhibited arginine uptake, lysine being most potent with a K_i of 1.2 millimolar. In the presence of free ATP, but not of its Mg-complex, uptake of arginine was drastically enhanced and a linear function of its concentration up to 16 millimolar. The nonhydrolyzable adenylyl imidodiphosphate, but no other nucleotide tested, could substitute for ATP. Therefore, it is suggested that this process does not require energy and does not involve the tonoplast ATPase. The ATP-dependent arginine uptake was strongly inhibited by p-chloromercuriphenylsulfonic acid. Furthermore, hydrophobic amino acids were inhibitory (I₅₀ phenylalanine 1 millimolar). Similar characteristics were observed for the uptake of aspartic acid. However, rates of ATP-stimulated aspartic acid transport were 10-fold lower as compared to arginine transport. Uptake of aspartate in the absence of ATP was negligible.     

CHANGES IN THE LEVEL OF ARGININE PHOSPHATE FOLLOWING FERTILIZATION OF ECHINODERM EGGS*

The content of arginine phosphate was measured following fertilization of sea-urchin eggs and starfish oocytes. In sea-urchin eggs, a rise in the level of arginine phosphate occurred within 2 min after insemination: this was not accompanied by any detectable alteration in the level of ATP. On the other hand, the level of arginine phosphate in starfish oocytes did not change on fertilization.     

Locomotory,ventilatory and metabolic responses of the subterranean Stenasellus virei (Crustacea,Isopoda) to severe hypoxia and subsequent recovery

The locomotory and ventilatory activities and the intermediary and energy metabolism modifications of the hypogean aquatic isopod crustacean Stenasellus virei were investigated in severe hypoxia (Po₂ < 0.03 kPa) and subsequent recovery. The aims of this study were i) to determine why the subterranean species displayed a greater tolerance of hypoxia than numerous other epigean crustaceans, ii) to confirm previous results obtained with four hypogean and epigean crustaceans, iii) to compare the responses to severe hypoxia in hypogean amphipods and isopods, and iv) to better understand the ecological problems of the hypogean organisms survival in subterranean habitats. S. virei responded to experimental long-term, severe hypoxia with classical anaerobic metabolism mainly characterized by a decrease in adenosine triphosphate (ATP) and phosphagen, utilization of glycogen and glutamate, and accumulation of lactate and alanine. Lactate was also largely excreted by this organism, which is unusual for crustaceans in general. Compared to most other epigean crustaceans, the isopod S. virei showed high amounts of stored glycogen and arginine phosphate. These differences in glycogen and phosphagen stores, and the ability to reduce energetic expenditures linked to locomotion and ventilation, extended the survival of S. virei under experimental anaerobiosis. During recovery, the isopod S. virei showed a higher capacity for glyconeogenesis from lactate and a faster and total replenishment of ATP and arginine phosphate levels than epigean crustaceans. Data concerning responses to hypoxia and subsequent recovery in S. virei are similar to those previously obtained with two other hypogean amphipods, except that this isopod did not synthesize succinate in anaerobiosis.     

Carbamoyl phosphate synthetase subunit Cpa1 interacting with Dut1, controls development,arginine biosynthesis,and pathogenicity of Colletotrichum gloeosporioides

Carbamoyl phosphate synthetase is involved in arginine biosynthesis in many organisms. In this study, we investigate the biological function of Cpa1, a small subunit of carbamoyl phosphate synthetase of Colletotrichum gloeosporioides. The deletion of the CPA1 gene affected vegetative growth, arginine biosynthesis, and fungal pathogenicity. Genetic complementation with native CPA1 fully recovered all these defective phenotypes. We observed that Cpa1-RFP fusion protein is localized at the mitochondria, which is consistent with Cpa2, a large subunit of carbamoyl phosphate synthetase. We identified the proteins that interact with Cpa1 by using the two-hybrid screen approach, and we showed that Dut1 interacts with Cpa1 but without Cpa2 in vivo. Dut1 is dispensable for hyphal growth, appressorial formation, and fungal pathogenicity. Interestingly, the Dut1-Cpa1 complex is localized at the mitochondria. Further studies showed that Dut1 regulates Cpa1-Cpa2 interaction in response to arginine. In summary, our studies provide new insights into how Cpa1 interacts with its partner proteins to mediate arginine synthesis.     

Purification and properties of adductor muscle phosphofructokinase from the oyster, Crassostrea virginica. The aerobic/anaerobic transition: role of arginine phosphate in enzyme control.

Phosphofructokinase from oyster (Crassostrea virginica) adductor muscle occurs in a single electrophorectic form at an activity of 8.1 mumol of product formed per minute per gram wet weight. The enzyme was purified to homogeneity by a novel method involving extraction in dilute ethanol and subsequent precipitation with polyethylene glycol. Oyster adductor phosphofructokinase has a molecular weight of 3400000 +/- 20000 as measured by Sephadex gel chromatography. Mg2+ or Mn2+ can satisfy the divalent ion requirement while ATP, GTP, or ITP can serve as phosphate donors for the reaction. Oyster adductor phosphofructokinase displays hyperbolic saturation kinetics with respect to all substrates (fructose 6-phosphate, ATP, and Mg2+) at either pH 7.9 OR PH 6.8. The Michaelis constant for fructose 6 phosphate at pH 6.8, the cellular pH of anoxic oyster tissues, is 3.5 mM. In the presence of AMP, by far the most potent activator and deinhibitor of the enzyme, this drops to 0.70 mM. Many traditional effectors of phosphofructokinase including citrate, NAD(P)H,Ca2+, fructose 1,6-bisphosphate, 3-phosphoglycerate, ADP, and phosphoenolpyruvate do not alter enzyme activity when tested at their physiological concentrations. Monovalent ions (K +, NH4+) are activators of the enzyme. ATP and arginine phosphate are the only compounds found to inhibit the adductor enzyme. The inhibitory action of both can be reversed by physiological concentrations of AMP(0.2- 1.0mM) and to a lesser extent by high concentrations of Pi (20 mM) and adenosine 3' :5'-monophosphate (0.1 mM). The two inhibitors exhibit very different pH versus inhibition profiles. The Ki (ATP) decreases from 5.0 mM to 1.3 mM as the pH decreases from 7.9 to 6.8, whereas the Ki for arginine phosphate increases from 1.3 mM to 4.5 mM for the same pH drop. Of all compounds tested, only AMP, within its physiological range, activated adductor phosphofructokinase significantly at low pH values. The kinetic data support the proposal that arginine phosphate, not ATP or citrate, is the most likely regulator of adductor phosphofructokinase in vivo under aerobic, high tissue pH, conditions. In anoxia, the depletion of arginine phosphate reserves and the increase in AMP concentrations in the tissue, coupled with the increase in the Ki for arginine phosphate brought about by low pH conditions, serves to activate phosphofructokinase to aid maintenance of anaerobic energy production.     

Improved method for expression and isolation of the Mycoplasma hominis arginine deiminase from the recombinant strain of Escherichia coli

Arginine deiminase is a promising anticancer drug active against melanoma, hepatocarcinoma and other tumors. Recombinant strains of Escherichia coli that express arginine deiminase from pathogenic bacteria Mycoplasma have been developed. However, production costs of heterologous arginine deiminase are high due to use of an expensive inducer and extraction buffer, as well as using diluted culture for enzyme induction. We report on a new advanced protocol for Mycoplasma hominis arginine deiminase expression, extraction and renaturation. The main improvements include manipulation with dense suspensions of E. coli, use of lactose instead of isopropyl β-d-1-thiogalactopyranoside as an inducer and a cheaper but not less efficient buffer for solubilization of arginine deiminase inclusion bodies. In addition, supplementation of the storage culture medium with glucose and substrate (arginine) significantly stabilized the recombinant arginine deiminase producer. Homogenous preparations of recombinant arginine deiminase were obtained using anion-exchange and hydrophobic chromatography. The purified enzyme retained a specific activity of 30–34 U/mg for 12 months when stored at 4 °C in 20 mM sodium phosphate buffer pH 7.2 containing 1 M NaCl.     

Engineering the Genotype of Acinetobacter sp. Strain ADP1 To Enhance Biosynthesis of Cyanophycin

To study the importance of arginine provision and phosphate limitation for synthesis and accumulation of cyanophycin (CGP) in Acinetobacter sp. strain ADP1, genes encoding the putative arginine regulatory protein (argR) and the arginine succinyltransferase (astA) were inactivated, and the effects of these mutations on CGP synthesis were analyzed. The inactivation of these genes resulted in a 3.5- or 7-fold increase in CGP content, respectively, when the cells were grown on glutamate. Knockout mutations in both genes led to a better understanding of the effect of the addition of other substrates to arginine on CGP synthesis during growth of the cells of Acinetobacter sp. strain ADP1. Overexpression of ArgF (ornithine carbamoyltransferase), CarA-CarB (small and large subunits of carbamoylphosphate synthetase), and PepC (phosphoenolpyruvate carboxylase) triggered synthesis of CGP if amino acids were used as a carbon source whereas it was not triggered by gluconate or other sugars. Cells of Acinetobacter sp. strain ADP1, which is largely lacking genes for carbohydrate metabolism, showed a significant increase in CGP contents when grown on mineral medium supplemented with glutamate, aspartate, or arginine. The Acinetobacter sp. ΔastA(pYargF) strain is unable to utilize arginine but synthesizes more arginine, resulting in CGP contents as high as 30% and 25% of cell dry matter when grown on protamylasse or Luria-Bertani medium, respectively. This recombinant strain overcame the bottleneck of the costly arginine provision where it produces about 75% of the CGP obtained from the parent cells grown on mineral medium containing pure arginine as the sole source of carbon. Phosphate starvation is the only known trigger for CGP synthesis in this bacterium, which possesses the PhoB/PhoR phosphate regulon system. Overexpression of phoB caused an 8.6-fold increase in CGP content in comparison to the parent strain at a nonlimiting phosphate concentration.     

Influence of Carbohydrate Starvation and Arginine on Culturability and Amino Acid Utilization of Lactococcus lactis subsp. lactis

Two strains of Lactococcus lactis subsp. lactis were used to determine the influence of lactose and arginine on viability and amino acid use during carbohydrate starvation. Lactose provided energy for logarithmic-phase growth, and amino acids such as arginine provided energy after carbohydrate exhaustion. Survival time, cell numbers, and ATP concentrations increased with the addition of arginine to the basal medium. By the onset of lactose exhaustion, the concentrations of glycine-valine and glutamate had decreased by as much as 67% in L. lactis ML3, whereas the serine concentration increased by 97% during the same period. When no lactose was added, the concentrations of these amino acids remained constant. Similar trends were observed for L. lactis 11454. Without lactose or arginine, L. lactis ML3 was nonculturable on agar but was viable after 2 days, as measured by fluorescent viability stains and intracellular ATP levels. However, L. lactis 11454 without lactose or arginine remained culturable for at least 14 days. These data suggest that lactococci become viable but nonculturable in response to carbohydrate depletion. Additionally, these data indicate that amino acids other than arginine facilitate the survival of L. lactis during carbohydrate starvation.     

Dependence of Streptococcus lactis phosphate transport on internal phosphate concentration and internal pH.

Uptake of phosphate by Streptococcus lactis ML3 proceeds in the absence of a proton motive force, but requires the synthesis of ATP by either arginine or lactose metabolism. The appearance of free Pi internally in arginine-metabolizing cells corresponded quantitatively with the disappearance of extracellular phosphate. Phosphate transport was essentially unidirectional, and phosphate concentration gradients of up to 10(5) could be established. Substrate specificity studies of the transport system indicated no preference for either mono- or divalent phosphate anion. The activity of the phosphate transport system was affected by the intracellular Pi concentration by a feedback inhibition mechanism. Uncouplers and ionophores which dissipate the pH gradient across the cytoplasmic membrane inhibited phosphate transport at acidic but not at alkaline pH values, indicating that transport activity is regulated by the internal proton concentration. Phosphate uptake driven by arginine metabolism increased with the intracellular pH with a pKa of 7.3. Differences in transport activity with arginine and lactose as energy sources are discussed.     

Polyphosphatase PPN1 of Saccharomyces cerevisiae: Switching of Exopolyphosphatase and Endopolyphosphatase Activities

The polyphosphatase PPN1 of Saccharomyces cerevisiae shows an exopolyphosphatase activity splitting phosphate from chain end and an endopolyphosphatase activity fragmenting high molecular inorganic polyphosphates into shorter polymers. We revealed the compounds switching these activities of PPN1. Phosphate release and fragmentation of high molecular polyphosphate prevailed in the presence of Co²⁺ and Mg²⁺, respectively. Phosphate release and polyphosphate chain shortening in the presence of Co²⁺ were inhibited by ADP but not affected by ATP and argininе. The polyphosphate chain shortening in the presence of Mg²⁺ was activated by ADP and arginine but inhibited by ATP.     

Dual Mechanism of Ion Permeation through VDAC Revealed with Inorganic Phosphate Ions and Phosphate Metabolites

In the exchange of metabolites and ions between the mitochondrion and the cytosol, the voltage-dependent anion channel (VDAC) is a key element, as it forms the major transport pathway for these compounds through the mitochondrial outer membrane. Numerous experimental studies have promoted the idea that VDAC acts as a regulator of essential mitochondrial functions. In this study, using a combination of molecular dynamics simulations, free-energy calculations, and electrophysiological measurements, we investigated the transport of ions through VDAC, with a focus on phosphate ions and metabolites. We showed that selectivity of VDAC towards small anions including monovalent phosphates arises from short-lived interactions with positively charged residues scattered throughout the pore. In dramatic contrast, permeation of divalent phosphate ions and phosphate metabolites (AMP and ATP) involves binding sites along a specific translocation pathway. This permeation mechanism offers an explanation for the decrease in VDAC conductance measured in the presence of ATP or AMP at physiological salt concentration. The binding sites occur at similar locations for the divalent phosphate ions, AMP and ATP, and contain identical basic residues. ATP features a marked affinity for a central region of the pore lined by two lysines and one arginine of the N-terminal helix. This cluster of residues together with a few other basic amino acids forms a “charged brush” which facilitates the passage of the anionic metabolites through the pore. All of this reveals that VDAC controls the transport of the inorganic phosphates and phosphate metabolites studied here through two different mechanisms.     

Modes of modifier action in E. coli aspartate transcarbamylase

The observed patterns for inhibition by CTP and succinate of equilibrium exchange kinetics with native aspartate transcarbamylase (E. coli) are consistent with an ordered substrate-binding system in which aspartate binds after carbamyl phosphate, and phosphate is released after carbamyl aspartate. ATP selectively stimulates Asp carbamyl-Asp exchange, but not carbamyl phosphate P_i. Initial velocity studies at 5 °, 15 °, and 35 °C were carried out, using modifiers as perturbants of the system. Modifiers alter the Hill n and S_0.5 for aspartate, most markedly at 15 °C but less so at the other temperatures. ATP does increase V under saturating substrate conditions, and substrate inhibition is observed for aspartate. ATP does not make the Hill n = 1 at any temperature. It is proposed that CTP and ATP act by separate mechanisms, not by simply perturbing in opposite directions the equilibrium for aspartate binding. ATP appears to act to increase the rate of aspartate association and dissociation, whereas CTP induces an intramolecular competitive effect in the 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.