Aspartate Group Research Articles

A regioselective tandem reduction — Wittig-Horner reaction involving the α-ester moiety of diethyl aspartate or glutamate

The α-ester group of N-protected diethyl aspartate ( 1a) or diethyl glutamate ( 1b) was selectively reduced using diisobutylaluminum hydride (DIBALH) in the presence of a lithium trialkylphosphonoacetate ( 2) to afford N-protected γ-amino-α,β-unsaturated dicarboxylates ( 4).

Long-Term Anaerobic Metabolism in Root Tissue (Metabolic Products of Pyruvate Metabolism).

The onset of anaerobiosis in barley root tissue (Hordeum vulgare L. cv Himalaya) results in the following metabolic responses. There are rapid increases in the levels of pyruvate, lactate, and ethanol. Malate and succinate concentrations increase over the first 12 h, after which they return to the levels found in oxygenated root tissue. Alanine concentration increases over the first 12 h, and this is matched by a corresponding decrease in aspartate. The initial stoichiometric decline in aspartate and increase in alanine suggests that the amino group of aspartate is conserved by transaminating pyruvate to alanine. Aspartate catabolism also probably provides the initial source of carbon for reduction to succinate under anoxic conditions. Under long-term anaerobiosis (>24 h), there is no further accumulation of any of the fermentative end products other than ethanol, which also represents the major metabolic end product during long-term anaerobiosis. Although a number of the enzymes involved in fermentative respiration have been found to be induced under anaerobic conditions, neither aspartate amino-transferase nor malate dehydrogenase is induced in barley root tissue. The observations suggest that the long-term adaptations to hypoxic conditions may be quite different than the more well-characterized short-term adaptations.

Effects of environmental anoxia on concentrations of free amino acids and kinetic properties of glutamate dehydrogenase in three body parts of Phascolosoma arcuatum (Sipuncula)

The body wall (BW), introvert cum retractor muscles (ICRM) and digestive tract (DT) of the sipunculid worm Phascolosoma arcuatum had different biochemical adaptations to anoxia with reference to amino acid metabolism. Although aspartate degradation occurred in all three body parts of the anoxic worm, alanine and glutamate accumulated only in the ICRM and DT, and not the BW. Hence, in the BW, the amino group of aspartate was not transferred to alanine during anoxia, but was possibly released as ammonia through glutamate. The kinetic properties of glutamate dehydrogenase (GDH) from the BW and ICRM were altered during anoxia to enhance the glutamate deaminating activity. Although alanine and glutamate were accumulated in the ICRM of the anoxic P. arcuatum, the decrease in aspartate content was unable to account for the increase. Results indicate that it was in the DT of the anoxic worm that glutamate was likely to act as an intermediate to shuttle the amino group between aspartate and alanine. The kinetic properties of the GDH from this body part of P. arcuatum were unaltered by anoxia.

Effect of sodium aspartate on the recovery of the rat heart from long-term hypothermic storage

We have investigated the reported ability of aspartate to enhance greatly the cardioprotective properties of the St. Thomas' Hospital cardioplegic solution after prolonged hypothermic storage. Rat hearts (n = 8 per group) were excised and subjected to immediate arrest with St. Thomas' Hospital cardioplegic solution (2 minutes at 4 degrees C) with or without addition of monosodium aspartate (20 mmol/L). The hearts were then immersed in the same solution for 8 hours (4 degrees C) before heterotopic transplantation into the abdomen of homozygous rats and reperfusion in vivo for 24 hours. The hearts were then excised and perfused in the Langendorff mode (20 minutes). Addition of aspartate to St. Thomas' Hospital cardioplegic solution gave a small but significant improvement in left ventricular developed pressure, which recovered to 82 +/- 3 mm Hg compared with 70 +/- 2 mm Hg in control hearts (p less than 0.05). However, coronary flow and high-energy phosphate content were similar in both groups. In subsequent experiments hearts (n = 8 per group) were excised, arrested (2 minutes at 4 degrees C) with St. Thomas' Hospital cardioplegic solution containing a 0, 5, 10, 20, 30, 40, or 50 mmol/L concentration of aspartate, stored for 8 hours at 4 degrees C, and then reperfused for 35 minutes. A bell-shaped dose-response curve was obtained, with maximum recovery in the 20 mmol/L aspartate group (cardiac output, 48 +/- 5 ml/min versus 32 +/- 5 ml/min in the aspartate-free control group; p less than 0.05). However, additional experiments showed that a comparable improvement could be achieved simply by increasing the sodium concentration of St. Thomas' Hospital cardioplegic solution by 20 mmol/L. Similarly, if sodium aspartate (20 mmol/L) was added and the sodium content of the St. Thomas' Hospital cardioplegic solution reduced by 20 mmol/L, no significant protection was observed when recovery was compared with that of unmodified St. Thomas' Hospital cardioplegic solution alone. In still further studies, hearts (n = 8 per group) were perfused in the working mode at either high (greater than 80 ml/min) or low (less than 50 ml/min) left atrial filling rates. Under these conditions, if functional recovery was expressed as a percentage of preischemic function, artifactually high recoveries could be obtained in the low-filling-rate group. In conclusion, assessment of the protective properties of organic additives to cardioplegic solutions requires careful consideration of (1) the consequences of coincident changes in ionic composition and (2) the characteristics of the model used for assessment.

Glucose analog inhibitors of glycogen phosphorylase: the design of potential drugs for diabetes

The T-state crystal structure of the glucose-phosphorylase b complex has been used as a model for the design of glucose analogue inhibitors that may be effective in the regulation of blood glucose levels. Modeling studies indicated room for additional atoms attached at the C1-beta position of glucose and some scope for additional atoms at the C1-alpha position. Kinetic parameters were determined for alpha-D-glucose: Ki = 1.7 mM, Hill coefficient n = 1.5, and alpha (synergism with caffeine) = 0.2. For beta-D-glucose, Ki = 7.4 mM, n = 1.5, and alpha = 0.4. More than 20 glucose analogues have been synthesized and tested in kinetic experiments. Most were less effective inhibitors than glucose itself and the best inhibitor was alpha-hydroxymethyl-1-deoxy-D-glucose (Ki = 1.5 mM, n = 1.3, alpha = 0.4). The binding of 14 glucose analogues to glycogen phosphorylase b in the crystal has been studied at 2.4-A resolution and the structure have been refined to crystallographic R values of less than 0.20. The kinetic and crystallographic studies have been combined to provide rationalizations for the apparent affinities of glucose and the analogues. The results show the discrimination against beta-D-glucose in favor of alpha-D-glucose is achieved by an additional hydrogen bond made in the alpha-glucose complex through water to a protein group and an unfavorable environment for a polar group in the beta pocket. The compound alpha-hydroxymethyl-1-deoxy-D-glucose has an affinity similar to that of glucose and makes a direct hydrogen bond to a protein group. Comparison of analogues with substituent atoms that have flexible geometry (e.g., 1-hydroxyethyl beta-D-glucoside) with those whose substituent atoms are more rigid (e.g., beta-azidomethyl-1-deoxyglucose or beta-cyanomethyl-1-deoxyglucose) indicates that although all three compounds make similar polar interactions with the enzyme, those with more rigid substituent groups are better inhibitors. In another example, alpha-azidomethyl-1-deoxyglucose was a poor inhibitor. In the crystal structure the compound made several favorable interactions with the enzyme but bound in an unfavorable conformation, thus providing an explanation for its poor inhibition. Attempts to utilize a contact to a buried aspartate group were partially successful for a number of compounds (beta-aminoethyl, beta-mesylate, and beta-azidomethyl analogues). The beta pocket was shown to bind gentiobiose (6-O-beta-D-glucopyranosyl-D-glucose), indicating scope for binding of larger side groups for future studies.

Refined structure of porcine pepsinogen at 1·8 Å resolution

The molecular structure of porcine pepsinogen at 1.8 Å resolution has been determined by a combination of molecular replacement and multiple isomorphous phasing techniques. The resulting structure was refined by restrained-parameter least-squares methods. The final R factor (= Σ|| F o |-| F c ||/ Σ| F o |) is 0.164 for 32,264 reflections with I ≥ σ( I) in the resolution range of 8.0 to 1.8 Å. The model consists of 2785 protein atoms in 370 residues, a phosphoryl group on Ser68 and 238 ordered water molecules. The resulting molecular stereochemistry is consistent with a well-refined crystal structure with co-ordinate accuracy in the range of 0.10 to 0.15 Å for the well-ordered regions of the molecule (B < 15 A ̊ 2) . For the enzyme portion of the zymogen, the root-mean-square difference in C α atom co-ordinates with the refined porcine pepsin structure is 0.90 Å (284 common atoms) and with the C α atoms of penicillopepsin it is 1.63 Å (275 common atoms). The additional 44 N-terminal amino acids of the prosegment (Leu1p to Leu44p, using the letter p after the residue number to distinguish the residues of the prosegment) adopt a relatively compact structure consisting of a long β-strand followed by two approximately orthogonal α-helices and a short 3 10-helix. Intimate contacts, both electrostatic and hydrophobic interactions, are made with residues in the pepsin active site. The N-terminal β-strand, Leu1p to Leu6p, forms part of the six-stranded β-sheet common to the aspartic proteinases. In the zymogen the first 13 residues of pepsin, Ile1 to Glu13, adopt a completely different conformation from that of the mature enzyme. The C α atom of Ile1 must move ~44 Å in going from its position in the inactive zymogen to its observed position in active pepsin. Electrostatic interactions of Lys36pN and hydrogen-bonding interactions of Tyr37pOH and Tyr90H with the two catalytic aspartate groups, Asp32 and Asp215, prevent substrate access to the active site of the zymogen. We have made a detailed comparison of the mammalian pepsinogen fold with the fungal aspartic proteinase fold of penicillopepsin, used for the molecular replacement solution. A structurally derived alignment of the two sequences is presented.

The fidelity of protein synthesis: can mischarging by aspartyl-tRNA Asp synthetase lead to the formation of isoaspartyl residues in proteins?

We have tested the hypotesis that isoaspartic acid residues in proteins can arise via errors that occur during protein synthesis. One such error involves a mischarging step in which the aspartic acid side-chain β-carboxyl group is linked to the tRNA Asp instead of the main chain α-carboxyl group. If this altered Asp-tRNA Asp is a substrate for the ribosomal elongation reactions, a polypeptide will be made with an isoaspartic acid, or β-linkage, in which the peptide chain is branched at the side chain of the aspartic acid residue. Using an ammonium sulfate fraction of aspartyl-tRNA Asp synthetase from Escherichia coli and [ 3H]aspartic acid, we have prepared [ 3H]aspartyl-tRNA Asp complexes and directly analyzed the linkage of the [ 3H]aspartate to the tRNA by identifying the products of ammonolysis. Normal attachment of the α-carboxyl group of aspartate to the tRNA produces [ 3H]isoasparagine, while the mischarging reaction leads to [ 3H]asparagine formation after ammonolysis. We have separated [ 3H]isoasparagine from [ 3H]asparagine and found an upper limit of 1 asparagine per 10000 isoasparagines. These results show that the bacterial aminoacyl-tRNA synthetase can very accurately distinguish between the α- and β-carboxyl groups of aspartic acid and suggest that only a very small fraction of the isoaspartic acid residues found to occur in cellular proteins may be the result of mischarging steps.

A single amino acid substitution in lactate dehydrogenase improves the catalytic efficiency with an alternative coenzyme

Using site-directed mutagenesis, the NADH-linked lactate dehydrogenase from Bacillus stearothermophilus has been specifically altered at a single residue to shift the coenzyme specificity towards NADPH. The single change is at position 53 in the amino acid sequence where a conserved aspartate has been replaced by a serine. This substitution was made to reduce steric hindrance on binding of the extra phosphate group of NADPH and to remove the negative charge of the aspartate group. The resultant mutant enzyme is 20 times more catalytically efficient than the wild-type enzyme with NADPH.

Reaction of Woodward's reagent K with D-xylose isomerases. Modification of an active site carboxylate residue.

D-Xylose isomerases from Streptomyces violaceoruber, Streptomyces sp., Lactobacillus xylosus, Lactobacillus brevis and Bacillus coagulans were rapidly inactivated by Woodward's reagent K. Second-order rate constants in the absence of ligands, at pH 6.0 and 25 degrees C, were 41, 36, 22, 95 and 26 M-1.min-1 respectively. Spectral analysis at 340 nm revealed that inactivation was correlated with modification of five, six, two, three and six carboxylate residues per monomer respectively. In the presence of protecting ligands, modification of one carboxylate group was prevented. The results support the idea of an active site glutamate or aspartate group that may contribute to the catalytic activity of all these D-xylose isomerases.

How do serine proteases really work?

Recent advances in genetic engineering have led to a growing acceptance of the fact that enzymes work like other catalysts by reducing the activation barriers of the corresponding reactions. However, the key question about the action of enzymes is not related to the fact that they stabilize transition states but to the question to how they accomplish this task. This work considers the catalytic reaction of serine proteases and demonstrates how one can use a combination of calculations and experimental information to elucidate the key contributions to the catalytic free energy. Recent reports about genetic modifications of the buried aspartic group in serine proteases, which established the large effect of this group (but could not determine its origin), are analyzed. Two independent methods indicate that the buried aspartic group in serine proteases stabilizes the transition state by electrostatic interactions rather than by alternative mechanisms. Simple free energy considerations are used to eliminate the double proton-transfer mechanism (which is depicted in many textbooks as the key catalytic factor in serine proteases). The electrostatic stabilization of the oxyanion side of the transition state is also considered. It is argued that serine proteases and other enzymes work by providing electrostatic complementarity to the changes in charge distribution occurring during the reactions they catalyze.

Incorporation of histidine into the pyrimidine moiety of thiamin in Saccharomyces cerevisiae

To investigate the incorporation of histidine into the pyrimidine moiety of thiamin in eukaryotes, Saccharomyces cerevisiae was grown in a synthetic medium in the presence of 15N- or 14C-labeled histidine. Two 15N-atoms of DL-[1,3-15N2]histidine were incorporated into the N-3 and amino-N atom at C-4 of pyrimidine. Furthermore, incorporation of the 15N-amino group of aspartate, the origin of the N-1 of histidine, into the N-3 of pyrimidine shows that N-3 and the amino-N atom at C-4 of pyrimidine are derived from N-1 and N-3 of histidine, respectively. In contrast, the 15N atom of DL-[amino-15N]histidine was not incorporated into the molecule, whereas L-[2-14C]histidine was incorporated directly into the pyrimidine. We conclude that N-1, C-2, and N-3 of histidine are the origins of the N-3, C-4, and amino-N at C-4 of the pyrimidine in thiamin synthesized by S. cerevisiae.

The role of an active site histidine in the catalytic mechanism of aspartate transcarbamoylase.

Although the allosteric enzyme aspartate transcarbamoylase from Escherichia coli has been the focus of numerous physical and enzymological studies for over 2 decades, the catalytic mechanism is still poorly understood. There has been much speculation regarding the role of a conserved histidine residue at position 134 which recent crystallographic studies have implicated in the catalytic mechanism as a general acid or as a general base. We have used a combination of site-directed mutagenesis, 13C-isotope incorporation, and high field NMR to probe the role of His134 in the catalytic mechanism of aspartate transcarbamoylase. By comparing the wild-type catalytic trimer with that from a partially active mutant in which His134 is replaced by alanine, we have assigned the 13C resonance for His134 in the wild-type enzyme. This residue is shown to have a pK less than 6, indicating that the imidazole ring is unprotonated at pH values optimal for enzymatic activity (pH 8.0). This result eliminates the possibility of His134 participating as a general acid in the carbamoyl transfer mechanism. Since the crystallographic studies indicate that His134 is close enough to hydrogen-bond to the carbonyl of the liganded bisubstrate analog N-(phosphonacetyl)-L-aspartate, the imidazole ring would be oriented as to make it unlikely that the N1 lone pair of electrons could participate in general base catalysis. Moreover, if His134 is implicated in base catalysis, we would have expected a much greater loss of activity upon its replacement by alanine. Perhaps the role of His134 is merely to help position the carbonyl group of carbamoyl phosphate for nucleophilic attack by the alpha-amino group of aspartate.

Mutations in the active site of Escherichia coli phosphofructokinase.

The enzyme-catalysed transfer of a phosphoryl group from ATP is an important reaction in a wide variety of biological processes. We demonstrate here the essential function of an aspartate group in the catalysis of phosphoryl transfer by Escherichia coli phosphofructokinase, and the minor role of an arginine residue. We have used oligonucleotide-directed mutagenesis to replace two amino-acid residues which X-ray analysis has shown to be close to the transferred phosphoryl group and we have analysed the forward and back reactions of the mutant enzymes by steady-state kinetics. Changing Asp 127 to Ser reduced the turnover number by a factor of 18,000 in the forward direction and 3,100 in the back reaction, and the Michaelis constant for fructose 1,6-bisphosphate in the reverse reaction by a factor of 45. This shows that this aspartate is a key residue in the rate enhancement by the enzyme, probably acting as a base in the reaction mechanism, and that it also destabilizes the product complex. Changing Arg 171 to Ser reduced the turnover numbers by about 3.4, showing that this arginine has only a minor effect on the catalysis.

Protonated state of methotrexate, trimethoprim, and pyrimethamine bound to dihydrofolate reductase

13C nuclear magnetic resonance (NMR) of methotrexate, trimethoprim and pyrimethamine enriched 90% with 13C at C2 has provided a sensitive means of detecting the state of protonation of the heterocyclic rings of these inhibitors. In each case, protonation of N1 causes an upfield movement of the chemical shift of C2 by more than 6 ppm. By this method it has been shown that, at pH values up to 9.2, methotrexate is bound to bovine liver dihydrofolate reductase with N1 of the inhibitor protonated, just as in the case of the complex with reductase from Streptococcus faecium and Lactobacillus casei. Furthermore, trimethoprim bound to reductase from any of the three sources, and pyrimethamine bound to either of the bacterial reductases also have N1 protonated even at pH values up to 10. This implies that in all cases there is a strong interaction between protonated N1 of the inhibitor and the carboxylate group of the active site aspartate or glutamate. In every case p K a of the bound inhibitor is increased by several units, a finding in accord with crystallographic evidence that inhibitor bound to L. casei reductase is in a hydrophobic environment and that N1 is not hydrogen-bonded to water. It was confirmed by titration of protein fluorescence that trimethoprim has greater affinity for bacterial reductase than for vertebrate (bovine) reductase, and that this selectivity is more marked in ternary complexes in which NADPH is also bound to the active site. However, the data cited above indicate that this difference in affinities is not due to a weaker ionic interaction between protonated N1 of trimethoprim and the bovine enzyme. Instead, binding of the trimethoprim side chain to hydrophobic sites on the enzyme must provide less binding energy in the case of the mammalian enzyme.

New Cholesteric and Nematic Lyotropic Mesophases from Di-Sodium N-Lauroyl-Aspartate

Abstract Lyotropic liquid crystals are prepared from racemic and optically active di-sodium N-lauroyl aspartate (SNLA). The optically active levo enantiomer is a suitable amphiphile for obtaining cholesteric lyotropic mesophases. In the present work, different types of mesophases, including cholesteric and nematic, were prepared and their type I or II magnetic behavior characterized by deuterium and 23Na NMR. The polarizing microscope textures were investigated, particularly with regards to magnetic field effects upon the samples. The usual chevron pattern was obtained for particular sample orientations and several characteristic effects were observed for the type I cholesteric lyomesophase in the first few minutes following removal of the sample from the magnetic field. Although the head group of di-sodium N-lauroyl aspartate possesses two negative charges, compositions are not too different from those reported for other amphiphile lyotropic systems.

The active site of lysosomal sphingomyelinase: evidence for the involvement of hydrophobic and ionic groups.

The natural substrate for sphingomyelinase contains hydrophobic and polar moieties. In this study, we have employed pH rate studies and examined hydrophobic compounds and phosphorylated esters for their effect on sphingomyelinase activity in an attempt to determine some of the kinetic properties of this enzyme. Sphingomyelinase, purified from human placentae, undergoes noncompetitive inhibition by octylglucoside and Nonidet P-40, two nonionic detergents containing terminal octyl groups. The effect of these detergents at the hydrophobic binding site is somewhat different from that of Triton X-100, which contains an isooctyl terminal group, and this may serve to identify a structural basis for the effects. Sphingomyelinase activity is also modulated by several nucleotides. Inhibition by 5'-adenosine monophosphate (5'-AMP) is also noncompetitive. Other nucleotide monophosphates (such as 5'-uridine monophosphate (5'-UMP), 5'-cytidine monophosphate (5'-CMP), 2'-adenosine monophosphate (2'-AMP), and 3'-adenosine monophosphate (3'-AMP) and phosphorylated intermediates (such as phosphorylcholine, phosphorylethanolamine and hexose phosphates) have a lower inhibitory effect. The data suggest that the inhibition by 5'-AMP involves the combined effect of the phosphate group and the purine ring, structural requirements which may also be satisfied by bis(4-methylumbelliferyl)phosphate, a synthetic enzyme substrate. Studies of pH rate indicate that the maximal velocity for the hydrolysis of sphingomyelin is independent of pH over the range 3.5-6.2 while the Km value shows a pH dependence. The Km value is lowest from pH 4.0-5.2 and rises at pH values outside this range. The log Vmax/Km and pKm relationships, when plotted as a function of pH, have been used to identify the dissociation constants for the binding of sphingomyelin by the enzyme. These occur at pK values of 4.1 and 5.5. The activity of sphingomyelinase is also reduced when the enzyme is photooxidized in the presence of methylene blue or rose bengal and carbamylated by diethylpyrocarbonate (DEPC). These results are interpreted to show that 1). the enzyme contains a hydrophobic binding site which involves linear aliphatic moieties containing at least eight carbon atoms; 2) two ionic groups are involved in formation of the enzyme substrate complex, one of which is presumed to be the carboxylate group of aspartate or glutamate (represented by pK 4.1) and the second may be the protonated imidazolium group of histidine (represented by pK 5.5); and 3) since the maximal velocity shows no pH dependence, the interactions involving the hydrophobic and ionic groups affect only the binding of the substrate to the enzyme and formation of the enzyme-substrate complex.

Some stereochemical features of aspartate beta-decarboxylase.

Aspartate beta-decarboxylase catalyzes abortive decarboxylation/transamination of [2-3H]aspartate with at least 17% internal transfer of tritium to the pro-S position at C-4' of the resulting pyridoxamine phosphate. In the normal beta-decarboxylation reaction, at least 1.06% of the tritium from the alpha-position of aspartate appears in the product alanine. The enzyme catalyzes slow hydrogen exchange from the beta-position of alanine but not aspartate. The replacement of the beta-carboxyl group of aspartate by hydrogen occurs in an inversion mode. These results are interpreted in terms of a two-base mechanism.

15N2 Incorporation and metabolism in the lichen Peltigera aphthosa Willd

The Nostoc in the cephalodia of the lichen Peltigera aphthosa Willd. fixed (15)N2 and the bulk of the nitrogen fixed was continuously transferred from it to its eukaryotic partners (a fungus and a green alga, Coccomyxa sp.). Kinetic studies carried out over the first 30 min, after exposure of isolated cephalodia to (15)N2, showed that highest initial (15)N2-labelling was into NH 4 (+) . After 12 min little further increase in the NH 4 (+) label occurred while that in the amide group of glutamine and in glutamate continued to increase. The (15)N-labelling of the amino group of glutamine and of aspartate increased more slowly, followed by an increase in the labelling of alanine. When total incorporation of (15)N-label was calculated, the overall pattern was found to be rather similar except that, throughout the experiment, the total (15)N incorporated into glutamate was about six times greater than that into the amide group of glutamine. Pulse chase experiments, in which (14)N2 was added to cephalodia previously exposed to (15)N2, showed that the NH 4 (+) pool rapidly became depleted of (15)N-label, followed by decreases in the labelling of glutamate, the amide group of glutamine and aspartate. The (15)N-labelling of alanine, however, continued to increase for a period. When isolated cephalodia were treated with L-methionine-SR-sulphoximine, an inhibitor of glutamine synthetase (EC 6.3.1.2), and azaserine, an inhibitor of glutamate synthase (EC 2.6.1.53), there was no detectable labelling in glutamine although the (15)N-labelling of glutamate increased unimpaired. On treating the cephalodia with amino-oxyacetate, an inhibitor of aminotransferase activity, the alanine pool decreased. Evidence was obtained that glutamine synthetase and glutamate synthase were located in the Nostoc, and that glutamate dehydrogenase (EC 1.4.1.4) and various amino-transferases were located in the cephalodial fungus. Possible implications of these findings are discussed.

Proton nuclear magnetic resonance evidence for the absence of a stable hydrogen bond between the active site aspartate and histidine residues of native subtilisins and for its presence in thiolsubtilisins.

The very low field proton nuclear magnetic resonance (1H NMR) found in aqueous solutions of serine proteases and their zymogens is characteristic of the hydrogen bond between the imidazolium and aspartate groups of the catalytic triad: Ser-His-Asp [Robillard, G., & Shulman, R. G. (1972) J. Mol. Biol. 71, 507--511]. According to 1H correlation NMR spectroscopic studies performed in 80/20 (v/v) H2O/2H2O, no such resonance is found in native subtilisins (even at -2 degrees C and pH 6.0), but it is present in thiolsubtilisins and in the phenylboronic acid derivatives of the serine enzymes. The resonance was not visible in the mercuric or carboxamidomethyl derivatives of the thiol enzymes or in the phenylboronic acid--serine enzyme complex if the serine enzyme was first acylated with phenylmethanesulfonyl fluoride. The histidine at the catalytic site of thiolsubtilisin carries a positive charge between pH 5.6 and 8.4, in accord with previous data in favor of a mercaptide--imidazolium ion pair at the catalytic site. The charge distribution (- + -) at the active site of thiolsubtilisin and in the phenylboronic acid derivatives of the serine enzymes resembles that in the tetrahedral transition state formed between a serine enzyme and its substrate. Therefore, the stable hydrogen bond (found in the thiol enzyme and in the phenylboronic acid derivative of the serine enzyme) should be more important during catalysis than in the substrate-free enzyme.

Autotrophic CO2 fixation in Chlorobium limicola. Evidence for the operation of a reductive tricarboxylic acid cycle in growing cells

Chlorobium limicola was grown on a mineral salts medium with CO2 as the main carbon source supplemented with specifically labeled 14C propionate and the incorporation of 14C into alanine (≙ intracellular pyruvate), aspartate (≙ oxaloacetate), and glutamate (≙ α-ketoglutarate) was studied in long term labeling experiments. During growth in presence of propionate 30% of the cell carbon were derived from propionate and 70% from CO2. Propionate was not oxidized to CO2. All three amino acids were found to be labeled. The labeling patterns indicate that propionate was assimilated via propionyl CoA, methylmalonyl CoA and succinyl CoA. When 1-14C propionate was the labeled precursor no radioactivity was found in the carboxyl group(s) of alanine, aspartate and glutamate, excluding the incorporation of propionate into the amino acids via succinate oxidation to fumarate. With 1-14C propionate preferentially aspartate (C-3) and glutamate (C-2) became labeled, with 2-14C propionate alanine (C-3) and glutamate (C-4). These findings indicate that propionate was incorporated into the amino acids via succinyl CoA, α-ketoglutarate, isocitrate, and citrate, followed by a si-type cleavage of citrate to oxaloacetate and acetyl CoA (or acetate). Similar experiments with U-14C acetate confirm these conclusions. Thus, all reactions of the proposed reductive tricarboxylic acid cycle could be demonstrated in autotrophically growing cells.