Glutamate-41 of Vibrio harveyi acyl carrier protein is essential for fatty acid synthase but not acyl-ACP synthetase activity

Bacterial acyl carrier protein (ACP) is a small, acidic, and highly conserved protein that supplies acyl groups for biosynthesis of a variety of lipid products. Recent modelling studies predict that residues primarily in helix II of Escherichia coli ACP (Glu-41, Ala-45) are involved in its interaction with the condensing enzyme FabH of fatty acid synthase. Using recombinant Vibrio harveyi ACP as a template for site-directed mutagenesis, we have shown that an acidic residue at position 41 is essential for V. harveyi fatty acid synthase (but not acyl-ACP synthetase) activity. In contrast, various replacements of Ala-45 were tolerated by both enzymes. None of the mutations introduced dramatic structural changes based on circular dichroism and native gel electrophoresis. These results confirm that Glu-41 of ACP is a critical residue for fatty acid synthase, but not for all enzymes that utilize ACP as a substrate.     

Multiple forms of beta-ketoacyl-acyl carrier protein synthetase in Escherichia coli.

Two forms of beta-ketoacyl-acyl carrier protein (ACP) synthetase (designated I and II) have been identified in extracts of Escherichia coli. Synthetase I corresponds to the condensing enzyme that was studied earlier (GREENSPAN, M.D., ALBERTS, A.W., and VAGELOS, P.R. (1969) J. Biol. Chem. 244, 6477-6485); synthetase II represents a new form of the enzyme. Synthetase II was isolated as a homogeneous protein. It differs from synthetase I in having a higher molecular weight (76,999 versus 66,000), a lower pH optimum (5.5 to 6.1 versus 7.2), and a greater resistance to denaturation by heat. Synthetase II is similar to synthetase I in that both are inactivated by iodoacetamide, and prior incubation of the enzymes with fatty acyl thioesters prevents the inhibitory effect of iodoacetamide. Both also react with a fatty acyl thioester to form an acyl-enzyme intermediate, and the latter reacts with malonyl-ACP to form a beta-ketoacyl thioester. Specificity studies indicated that synthetase II, like synthetase I, has similar affinities with saturated and cis unsaturated fatty acyl thioesters of ACP that are intermediates in the synthesis of saturated and unsaturated fatty acids, respectively. The two synthetases differ only with respect to reactivity with palmitoleyl thioesters: synthetase II has a lower Km and higher Vmax than synthetase I with palmitoleyl-ACP. This finding suggests that synthetase II functions specifically in the elongation of palmitoleyl-ACP to form cis-vaccenyl-ACP. An investigation of synthetases I and II in two classes of unsaturated fatty acid auxotrophs revealed that synthetase I is absent in one class, fabB. Addition of wild type synthetase I to fabB fatty acid synthetase, which synthesizes only saturated fatty acids, permitted this fatty acid synthetase to synthesize unsaturated fatty acids. These experiments indicate that synthetase I plays a critical role in the synthesis of unsaturated fatty acids.     

Purification and characterization of fatty acyl-acyl carrier protein synthetase from Vibrio harveyi.

A Vibrio harveyi enzyme which catalyzes the ATP-dependent ligation of fatty acids to acyl carrier protein (ACP) has been purified 6,000-fold to apparent homogeneity by anion-exchange, gel filtration, and ACP-Sepharose affinity chromatography. Purified acyl-ACP synthetase migrated as a single 62-kDa band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and as an 80-kDa protein by gel filtration under reducing conditions. Activity of the purified enzyme was lost within hours in the absence of glycerol and low concentrations of Triton X-100. Acyl-ACP synthetase exhibited Kms for myristic acid, ACP, and ATP of 7 microM, 18 microM, and 0.3 mM, respectively. The enzyme was specific for adenine-containing nucleotides, and AMP was the product of the reaction. No covalent acyl-enzyme intermediate was observed. Enzyme activity was stimulated up to 50% by iodoacetamide but inhibited > 80% by N-ethylmaleimide: inhibition by the latter was prevented by ATP and ACP but not myristic acid. Dithiothreitol and sulfhydryl-directed reagents also influenced enzyme size, activity, and elution pattern on anion-exchange resins. The function of acyl-ACP synthetase has not been established, but it may be related to the capacity of V. harveyi to elongate exogenous fatty acids by an ACP-dependent mechanism.     

Gamma-glutamylcysteine synthetase. Interactions of an essential sulfhydryl group

gamma-Glutamylcysteine synthetase (isolated from rat kidney) has one sulfhydryl group that reacts with 5,5'-dithiobis-(2-nitrobenzoate). This single exposed sulfhydryl group is not required for enzyme activity. The enzyme is potently inactivated by cystamine, which apparently interacts with a sulfhydryl group at the active site to form a mixed disulfide. 5,5'-Dithiobis-(2-nitrobenzoate) does not interact with the sulfhydryl group that reacts with cystamine. After the enzyme was 90% inactivated by reaction with cystamine, 3.4 mol of 5,5'-dithiobis-(2-nitrobenzoate) reacted per mol of enzyme, indicating that binding of cystamine exposes sulfhydryl groups which are apparently buried or unreactive in the native enzyme. L-Glutamate (but not D-glutamate or L-alpha-aminobutyrate) protected against inactivation by cystamine. In contrast, ATP enhanced the rate of inactivation by cystamine, and the apparent Km value for this effect is similar to that for ATP in the catalytic reaction. Studies on the structural features of cystamine that facilitate its interaction with the enzyme showed that selenocystamine, monodansylcystamine, and N-[2[2-aminoethyl)-dithio)ethyl]-4-azido-2-nitrobenzeneamine are also good inhibitors. Whereas S-(S-methyl)cysteamine-Sepharose does not interact with the enzyme (Seelig, G. F., and Meister, A. (1982) J. Biol. Chem. 257, 5092-5096), S-(S-methyl)cysteamine is a potent inhibitor; 1 mol of this compound completely inactivated 1 mol of enzyme. In the course of this work, a useful modification of the method for isolating this enzyme from kidney was developed.     

Effect of N-bromosuccinimide modification on dihydrofolate reductase from a methotrexate-resistant strain of Escherichia coli. Activity, spectrophotometric, fluorescence and circular dichroism studies.

When dihydrofolate reductase from a methotrexate-resistant strain of Escherichia coli B, MB 1428, is treated with approximately a 5 mol ratio of N-bromosuccinimide (NBS) to enzyme at pH 7.2 and assayed at the same pH, there is a 40% loss of activity due to the modification of 1 histidine residue and possibly 1 methionine residue before oxidation of tryptophan occurs. The initial modification is accompanied by a shift of the pH for maximal enzymatic activity from pH 7.2 to pH 5.5 Upon further treatment with N-bromosuccinimide, the activity is gradually reduced from 60 to 0% as tryptophan residues become oxidized. An NBS to enzyme mole ratio of approximately 20 results in 90% inactivation of the enzyme. When the enzyme is titrated with NBS in 6 M guanidine HCl, 5 mol of tryptophan react per mol of enzyme, a result in agreement with the total tryptophan content as determined by magnetic circular dichroism. The 40% NBS-inactivated sample posses full binding capacity for methotrexate and reduced triphosphopyridine nucleotide, and the Km values for dihydrofolate and TPNH are the same as for the native enzyme. After 90% inactivation, only half of the enzyme molecules bind methotrexate, and the dissociation constant for methotrexate is 40 nM as compared to 4 nM for native enzyme in solutions of 0.1 M ionic strength, pH 7.2 Also, TPNH is not bound as tightly to the modified enzyme-methotrexate complex as to the unmodified enzyme-methotrexate complex. Circular dichroism studies indicate the 90% NBS-inactivated enzyme has the same alpha helix content as the native enzyme but less beta structure, while the 40% inactivated enzyme is essentially the same as the native enzyme. Protection experiments were complicated by the fact that NBS reacts with the substrates and cofactors of the enzyme. Although protection of specific residues was not determined, it was clear that TPNH was partially protected from NBS reaction when bound to the enzyme, and the enzyme, and the enzyme was not inactivated by NBS until the TPNH had reacted.     

Preparation of fatty-acylated derivatives of acyl carrier protein using Vibrio harveyi acyl-ACP synthetase.

A simple two-step purification of Vibrio harveyi fatty acyl-acyl carrier protein (acyl-ACP) synthetase, which is useful for the quantitative preparation and analysis of fatty-acylated derivatives of ACP, is described. Acyl-ACP synthetase can be partially purified from extracts of this bioluminescent bacterium by Cibacron blue chromatography and Sephacryl S-300 gel filtration and is stable for months at -20 degrees C in the presence of glycerol. Incubation of ACP from Escherichia coli with ATP and radiolabeled fatty acids (6 to 16 carbons in length) in the presence of the enzyme resulted in quantitative conversion to biologically active acylated derivatives. The enzyme reaction can be monitored by a filter disk assay to quantitate levels of ACP or by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography to detect ACP in cell extracts. With its broad fatty acid chain length specificity and optimal activity in mild nondenaturing buffers, the soluble V. harveyi acyl-ACP synthetase provides an attractive alternative to current chemical and enzymatic methods of acyl-ACP preparation and analysis.     

Regulation of fatty acid synthetase activity. The 4'-phosphopantetheine hydrolase of rat liver.

The 4'-phosphopantetheine hydrolase of rat liver, partially purified by ammonium sulfate precipitation, catalyzes the hydrolysis of the prosthetic group 4'-phosphopantetheine from the holo-fatty acid synthetase. The two products of the action of this enzyme, 4'-phosphopantetheine and apo-fatty acid synthetase, were isolated by DEAE-cellulose chromatography and by chromatography on a Sepharose epsilon-aminocaproyl pantetheine column, respectively. The resultant apo-fatty acid synthetase was quantitated by immunoprecipitation and it was also converted to the holoprotein with a crude preparation of rat liver 4'-phosphopantetheine transferase. Quantitative determination of the hydrolase reaction product, 4'-phosphopantetheine, by amino acid analysis and microbiological assays confirmed the presence of 1 mol of this compound/mol of holo-fatty acid synthetase.     

Mechanism of mitochondrial carbamoyl-phosphate synthetase: synthesis and properties of active CO2, precursor of carbamoyl phosphate.

This paper demonstrates the formation of "active CO2" (CO2-P), a precursor of carbamoyl phosphate (CP), with frog liver carbamoyl-phosphate synthetase. Absence of ammonia is essential for the demonstration by pulse incubation with H14CO3- of CO2-P. Adenosine triphosphate (ATP) and acetylglutamate are required for the synthesis of CO2-P, which is highly unstable in aqueous solutions (t1/2 = 0.75 s at 24 degrees C at neutral pH). In the absence of ammonia, CO2-P attains rapidly a steady-state level, which depends on the concentration of ATP and HCO3-. The "apparent KM'S" are approximately equal to those found for the adenosine triphosphate (ATPase) activity of the enzyme. The maximum level of CO2-P is limited by the amount of enzyme, and approximates 4 mol of intermediate/mol of enzyme. The unprotonated form of ammonia seems to be the species reacting with CO2-P to produce CP. The reaction of CO2-P and NH3 is very fast (rate constant kn = 8 x 10(4) M-1 S-1) and does not consume free ATP. Therefore, the 2 mol of ATP necessary for CP synthesis binds or reacts with the enzyme and/or CO2 prior to reaction with NH3. The reaction of CO2-P with NH3 also takes place in acetone under conditions at which the enzyme is not active, suggesting little or no assistance from enzyme catalysis or that a part of the catalytic site is "frozen" by the solvent in the active conformation. In the light of these and other findings, a new scheme is proposed for the mechanism of frog liver carbamoyl-phosphate synthetase and some considerations are made on the chemical nature of the intermediate and on the possible evolutionary significance of the reaction of CO2-P with NH3 in acetone.     

Characterization of nonheme iron and reaction mechanism of bromoperoxidase in Corallina pilulifera

The properties of the nonheme iron of bromoperoxidase from Corallina pilulifera were studied. The enzyme lost its activity when reduced with formamidine-sulfinic acid and recovered it when oxidized by air. Incubation of the enzyme with ferric or ferrous ion-chelating agents indicated that its nonheme iron was ferric. Analyses of circular dichroism and proton NMR spectra suggested that the ferric ion tightly bound to cysteine, histidine, or tyrosine residues of the enzyme. The enzyme catalyzed Br--dependent catalase reactions to yield 1 mol of O2 from 2 mol of H2O2. No O2 evolution was observed when bromination reaction of monochlorodimedone occurred. From these results, together with previous knowledge of this enzyme, it was concluded that it activated bromide anion (Br-) to bromonium cation (Br+) using one molecule of H2O2, and this Br+OH- formed at the active site then decomposed another H2O2 to yield O2 in the absence of halogen acceptors (substrate). When substrate was present in the reaction mixture, it and H2O2 competitively reacted with the reaction intermediate (Br+OH-) to give brominated products.     

Site-directed mutagenesis of acyl carrier protein (ACP) reveals amino acid residues involved in ACP structure and acyl-ACP synthetase activity.

Acyl carrier protein (ACP) interacts with many different enzymes during the synthesis of fatty acids, phospholipids, and other specialized products in bacteria. To examine the structural and functional roles of amino acids previously implicated in interactions between the ACP polypeptide and fatty acids attached to the phosphopantetheine prosthetic group, recombinant Vibrio harveyi ACP and mutant derivatives of conserved residues Phe-50, Ile-54, Ala-59, and Tyr-71 were prepared from glutathione S-transferase fusion proteins. Circular dichroism revealed that, unlike Escherichia coli ACP, V. harveyi-derived ACPs are unfolded at neutral pH in the absence of divalent cations; all except F50A and I54A recovered native conformation upon addition of MgCl(2). Mutant I54A was not processed to the holo form by ACP synthase. Some mutations significantly decreased catalytic efficiency of ACP fatty acylation by V. harveyi acyl-ACP synthetase relative to recombinant ACP, e.g. F50A (4%), I54L (20%), and I54V (31%), whereas others (V12G, Y71A, and A59G) had less effect. By contrast, all myristoylated ACPs examined were effective substrates for the luminescence-specific V. harveyi myristoyl-ACP thioesterase. Conformationally sensitive gel electrophoresis at pH 9 indicated that fatty acid attachment stabilizes mutant ACPs in a chain length-dependent manner, although stabilization was decreased for mutants F50A and A59G. Our results indicate that (i) residues Ile-54 and Phe-50 are important in maintaining native ACP conformation, (ii) residue Ala-59 may be directly involved in stabilization of ACP structure by acyl chain binding, and (iii) acyl-ACP synthetase requires native ACP conformation and involves interaction with fatty acid binding pocket residues, whereas myristoyl-ACP thioesterase is insensitive to acyl donor structure.     

Inactivation of glutamine synthetases by an NAD:arginine ADP-ribosyltransferase

Glutamine synthetase from ovine brain has a critical arginine residue at the catalytic site (Powers, S. G., and Riordan, J.F. (1975) Proc. Natl. Acad. Sci. U.S. A. 72, 2616-2620). This enzyme is now shown to be a substrate for a purified NAD:arginine ADP-ribosyltransferase from turkey erythrocyte cytosol that catalyzes the transfer of ADP-ribose from NAD to arginine and purified proteins. The transferase catalyzed the inactivation of the synthetase in an NAD-dependent reaction; ADP-ribose and nicotinamide did not substitute for NAD. Agmatine, an alternate ADP-ribose acceptor in the transferase-catalyzed reaction, prevented inactivation of glutamine synthetase. MgATP, a substrate for the synthetase which was previously shown to protect that enzyme from chemical inactivation, also decreased the rate of inactivation in the presence of NAD and ADP-ribosyltransferase. Using [32P]NAD, it was observed that approximately 90% inactivation occurred following the transfer of 0.89 mol of [32P]ADP-ribose/mol of synthetase. The erythrocyte transferase also catalyzed the NAD-dependent inactivation of glutamine synthetase purified from chicken heart; 0.60 mol of ADP-ribose was transferred per mol of enzyme, resulting in a 95% inactivation. As noted with the ovine brain enzyme, agmatine and MgATP protected the chicken synthetase from inactivation and decreased the extent of [32P]ADP-ribosylation of the synthetase. These observations are consistent with the conclusion that the NAD:arginine ADP-ribosyltransferase modifies specifically an arginine residue involved in the catalytic site of glutamine synthetase. Although the transferase can use numerous proteins as ADP-ribose acceptors, some characteristics of this particular arginine, perhaps the same characteristics that are involved in its function in the catalytic site, make it a favored ADP-ribose acceptor site for the transferase.     

Purification and characterization of recombinant spinach acyl carrier protein I expressed in Escherichia coli

Expression of plant acyl carrier protein (ACP) in Escherichia coli at levels above that of constitutive E. coli ACP does not appear to substantially alter bacterial growth or fatty acid metabolism. The plant ACP expressed in E. coli contains pantetheine and approximately 50% is present in vivo as acyl-ACP. We have purified and characterized the recombinant spinach ACP-I. NH2-terminal amino acid sequencing indicated identity to authentic spinach ACP-I, and there was no evidence for terminal methionine or formylmethionine. Recombinant ACP-I was found to completely cross-react immunologically with polyclonal antibody raised to spinach ACP-I. Recombinant ACP-I was a poor substrate for E. coli fatty acid synthesis. In contrast, Brassica napus fatty acid synthetase gave similar reaction rates with both recombinant and E. coli ACP. Similarly, malonyl-coenzyme A:acyl carrier protein transacylase isolated from E. coli was only poorly able to utilize the recombinant ACP-I while the same enzyme from B. napus reacted equally well with either E. coli ACP or recombinant ACP-I. E. coli acyl-ACP synthetase showed a higher reaction rate for recombinant ACP-I than for E. coli ACP. Expression of spinach ACP-I in E. coli provides, for the first time, plant ACP in large quantities and should aid in both structural analysis of this protein and in investigations of the many ACP-dependent reactions of plant lipid metabolism.     

Yeast cystathionine beta-synthase reacts with L-allothreonine, a non-natural substrate, and L-homocysteine to form a new amino acid, 3-methyl-L-cystathionine

Our studies of the reaction mechanism of cystathionine beta-synthase from yeast (Saccharomyces cerevisiae) are facilitated by the spectroscopic properties of the pyridoxal phosphate coenzyme. The enzyme catalyzes the reaction of L-serine with L-homocysteine to form L-cystathionine through a series of pyridoxal phosphate intermediates. In this work, we explore the substrate specificity of the enzyme by use of substrate analogues combined with kinetic measurements under pre-steady-state conditions and with circular dichroism and fluorescence spectroscopy under steady-state conditions. Our results show that L-allothreonine, but not L-threonine, serves as an effective substrate. L-Allothreonine reacts with the pyridoxal phosphate cofactor to form a stable 3-methyl aminoacrylate intermediate that absorbs maximally at 446 nm. The rapid-scanning stopped-flow results show that the binding of L-allothreonine as the external aldimine is faster than formation of the 3-methyl aminoacrylate intermediate. The 3-methyl aminoacrylate intermediate reacts with L-homocysteine to form a new amino acid, 3-methyl-L-cystathionine, which was characterized by nuclear magnetic resonance spectroscopy. This new amino acid may be a useful analogue of L-cystathionine.     

Altered protein-chromophore interaction in dicyclohexylcarbodiimide-modified purple membrane sheets

Previous studies of N,N'-dicyclohexylcarbodiimide (DCCD)-modified bacteriorhodopsin (Renthal, R. et al. (1985) Biochemistry 24, 4275-4279) used reaction conditions (detergent micelles) that are not optimal for subsequent physical studies. The present work describes new conditions for reaction of bacteriorhodopsin with DCCD in intact purple membrane sheets in the presence of 4.5% (v/v) diethylether and light. Like the detergent reaction system, the reaction is light induced, incorporates approximately 1 mol [14C]DCCD per mol bacteriorhodospin, and results in a bleached chromophore. Peptide mapping indicates that the likely site of modification in intact membranes is identical to the site in the detergent reaction system: Asp 115. The retinal chromophore of DCCD-modified purple membrane has an absorbance maximum at 390 nm and very little induced circular dichroism. The retinal is easily extracted in hexane, yielding a 3:1 ratio of all-trans to 13-cis retinal. Borohydride reduces the retinal onto the protein within the 1-71 region of the amino acid sequence. These results suggest that Asp-115 is near the retinal binding cavity of bacteriorhodopsin. When DCCD reacts with Asp 115, retinal is displaced from its binding site.     

A comparative study of sulfhydryl groups required for the catalytic activity of gramicidin S synthetase and isoleucyl tRNA synthetase

The sulfhydryl groups required for the catalytic activity of gramicidin S synthetase of Bacillus brevis and Escherichia coli isoleucyl tRNA synthetase were compared. In gramicidin S synthetase 2(GS 2), about four sulfhydryl groups react rapidly with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) or N-ethylmaleimide (NEM), and are essential for gramicidin S formation in the presence of gramicidin S synthetase 1 (GS 1). These sulfhydryl groups are protected against DTNB and NEM reactions by the preincubation of GS 2 with amino acid substrates in the presence of ATP and MgCl2, like the sulfhydryl groups that react rapidly with DTNB or NEM and are required for the catalytic activity of GS 1 and isoleucyl tRNA synthetase. In GS 2, GS 1, and isoleucyl tRNA synthetase, the sulfhydryl group that reacts rapidly with NEM and is required for the catalytic activity is involved in the amino acid binding as a thioester. In isoleucyl tRNA synthetase, it is suggested that isoleucine may be transferred from the isoleucine thioester enzyme complex to tRNA by a mechanism similar to that proposed for gramicidin S synthetase.     

Reaction of tryptophanyl-tRNA synthetase from beef pancreas with periodate-oxidized ATP

Tryptophanyl-tRNA synthetase from beef pancreas reacts with periodate-oxidized ATP according to biphasic kinetics. A rapid phase involves two groups of the protein, presumably lysine side-chains. The slow phase corresponds to the reaction of a larger number of groups. The time-course of the partial losses of the ATP-PPi isotopic exchange and of the aminoacylation activities of the enzyme follow the labelling of the two fast-reacting groups. However, the ability of the enzyme to form a bis(tryptophanyladenylate)-enzyme complex is not lost after reaction of these two groups with the reagent. The affinity for ATP is also unaffected by this initial labelling of the protein, as seen from the Km values of this substrate in the ATP-PPi isotopic exchange reaction. These data suggest that, in this fast initial reaction, oxidized ATP reacts neither with specific ATP-binding groups of the enzyme nor with any major catalytic residue of the tryptophan-activation site. In contrast with this first step, the further slow labelling of lysine residues leads to a disappearance of the aminoacylation ability of the enzyme, while it does not further affect the ATP-PPi exchange activity. The behaviour of beef tryptophanyl-tRNA synthetase during derivatization with oxidized ATP is therefore at variance with that which has been described for the homologous E. coli enzyme.     

Studies on tyrosine residues in porcine muscle adenylate kinase. Circular dichroism spectra and chemical modification with tetranitromethane.

Substrate-induced conformational change of porcine muscle adenylate kinase (EC 2.7.4.3) is evidenced by a change in circular dichroism spectra in the near ultraviolet. In the absence of tryptophan in porcine muscle adenylate kinase, the spectral change may be assigned to a perturbation of tyrosine chromophore(s). The spectral change was specific for adenine nucleotide binding and was greater with ATP than with AMP. In the x-ray model, Tyr153 and Tyr154 are located at a hinge region of two domains which form a deep active site cleft and are therefore susceptible to conformational change on substrate binding. Adenylate kinase was treated with equimolar tetranitromethane. The yellow-colored product, separated from unmodified enzyme by substrate gradient elution on a phosphocellulose column, had about 1 mol of nitrotyrosine per mol of the enzyme by amino acid analysis and showed a slightly higher Km value than native enzyme for ADP (Km = 0.50 mM compared with 0.25 mM for native adenylate kinase). Spectrophotometric titration of nitroadenylate kinase gave pKa 8.4 for the dissociation constant of the nitrotyrosyl hydroxyl group. On binding ATP the pKa value increased to 9.0 while AMP binding caused very little change. By peptide mapping of the carboxypeptidase digestion product, 0.70 mol of nitro group per mol of adenylate kinase was detected on Tyr153 and a small amount of nitro group was also found on Tyr95. From these results it is proposed that Tyr153 is directly or indirectly involved in the binding of ATP.     

Tyrosyl-tRNA synthetase from the bovine liver. Isolation and physico-chemical properties

Tyrosyl-tRNA synthetase of beef liver has been isolated and its properties have been studied. Tyrosyl-tRNA synthetase is a structural dimer of alpha 2 type. Mr of the enzyme subunit is about 59 kDa. Km values for substrates have been determined and compared with kinetic properties of tyrosyl-tRNA synthetases from different sources. The polymorphism of tyrosyl-tRNA synthetase was studied. The enzyme was separated into two different forms by chromatography on phosphocellulose P 11. P1-form is active only in the amino acid activation reaction. This form is not due to the phosphorylation of the enzyme. The low molecular weight form (38 kDa) was also isolated. This form appeared due to the limited endogenic proteolysis of the main form and retained full activity in the aminoacylation reaction. Tyrosyl-tRNA synthetase from beef liver has non-specific affinity to rRNA-sepharose.     

Mixed-valence cytochrome oxidase-formate complex. A steady-state intermediate.

At neutral pH, formate binds to the haem a3 component of cytochrome c oxidase to give a complex that reacts differently from the non-liganded enzyme with reducing agents. Addition of sodium dithionite to the formate complex leads directly to the formation of the fully reduced species, whereas reduction with ascorbate/tetramethylenephenylene-diamine can lead to the production of a mixed-valence species. The stability of this mixed-valence form was studied, and the species appears to represent a 'steady-state' situation that is stable only in the presence of an excess of O2 and reducing equivalents. Characterization of the mixed-valence complex by electron paramagnetic resonance and magnetic circular dichroism reveals the presence of reduced low-spin haem a together with reduced detectable copper and high-spin ferric haem a3.     

Chemical modification of Salmonella typhimurium phosphoribosylpyrophosphate synthetase with 5'-(p-fluorosulfonylbenzoyl)adenosine. Identification of an active site histidine

Liquid chromatographic procedures have been developed for rapidly locating the site of reaction of chemical modification reagents with Salmonella typhimurium 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP) synthetase. The enzyme was reacted with the active site-directed reagent 5'-(p-fluorosulfonylbenzoyl)adenosine (FSBA). FSBA bound to the enzyme with an apparent KD of 1.7 +/- 0.4 mM. The enzyme was inactivated during the reaction, and a limiting stoichiometry of 1.2 mol of FSBA/mol of enzyme subunit corresponded to complete inactivation. Inclusion of ATP in the reaction protected the enzyme from inactivation and incorporation of the reagent. Inclusion of ribose 5-phosphate increased the rate of reaction of PRPP synthetase with FSBA. Amino acid analyses of acid hydrolysates of modified enzyme failed to detect any known FSBA-amino acid adducts. Tryptic digestion of 5'-(p-fluorosulfonylbenzoyl)-[3H]adenosine-modified enzyme at pH 7.0 yielded a single radioactive peptide. The peptide, TR-1, was subjected to combined V8 and Asp-N protease digestion, and a single radioactive peptide was isolated. This radioactive peptide yielded the sequence Asp-Leu-His-Ala-Glu, which corresponded to amino acid residues 128-132 in S. typhimurium PRPP synthetase. No radioactivity was associated with any of the phenylthiohydantoin-amino acid fractions, all of which were recovered in good yield. A majority of the radioactivity was found in the waste effluent (64%) and on the glass fiber filter loaded into the sequenator (23%). The lability of the modification and the sequence of this peptide indicate His130 as the site of reaction with FSBA.