Characterization of the acetate binding pocket in the Methanosarcina thermophila acetate kinase

Acetate kinase catalyzes the reversible magnesium-dependent synthesis of acetyl phosphate by transfer of the ATP gamma-phosphoryl group to acetate. Inspection of the crystal structure of the Methanosarcina thermophila enzyme containing only ADP revealed a solvent-accessible hydrophobic pocket formed by residues Val(93), Leu(122), Phe(179), and Pro(232) in the active site cleft, which identified a potential acetate binding site. The hypothesis that this was a binding site was further supported by alignment of all acetate kinase sequences available from databases, which showed strict conservation of all four residues, and the recent crystal structure of the M. thermophila enzyme with acetate bound in this pocket. Replacement of each residue in the pocket produced variants with K(m) values for acetate that were 7- to 26-fold greater than that of the wild type, and perturbations of this binding pocket also altered the specificity for longer-chain carboxylic acids and acetyl phosphate. The kinetic analyses of variants combined with structural modeling indicated that the pocket has roles in binding the methyl group of acetate, influencing substrate specificity, and orienting the carboxyl group. The kinetic analyses also indicated that binding of acetyl phosphate is more dependent on interactions of the phosphate group with an unidentified residue than on interactions between the methyl group and the hydrophobic pocket. The analyses also indicated that Phe(179) is essential for catalysis, possibly for domain closure. Alignments of acetate kinase, propionate kinase, and butyrate kinase sequences obtained from databases suggested that these enzymes have similar catalytic mechanisms and carboxylic acid substrate binding sites.     

Direct enzymic determination of acetate in tissue extracts in the presence of labile acetate esters

Methods are presented for the direct spectrophotometric and fluorometric determinations of acetate in tissue extracts using beef heart mitochondria acetyl-CoA synthetase. The methods eliminate the need for diffusion or distillation of the acetate from the extract and thereby minimize the danger of breakdown of labile tissue acetate esters, an occurrence which would result in an overestimation of acetate. Acetyl-CoA, acetylcarnitine, and N-acetyl-aspartate do not break down to acetate during the extraction procedure and do not interfere with the assay.     

Mechanisms of acetate formation and acetate activation in halophilic archaea

The halophilic archaea Halococcus (Hc.) saccharolyticus, Haloferax (Hf.) volcanii, and Halorubrum (Hr.) saccharovorum were found to generate acetate during growth on glucose and to utilize acetate as a growth substrate. The mechanisms of acetate formation from acetyl-CoA and of acetate activation to acetyl-CoA were studied. Hc. saccharolyticus, exponentially growing on complex medium with glucose, formed acetate and contained ADP-forming acetyl-CoA synthetase (ADP-ACS) rather than acetate kinase and phosphate acetyltransferase or AMP-forming acetyl-CoA synthetase. In the stationary phase, the excreted acetate was completely consumed, and cells contained AMP-forming acetyl-CoA synthetase (AMP-ACS) and a significantly reduced ADP-ACS activity. Hc. saccharolyticus, grown on acetate as carbon and energy source, contained only AMP-ACS rather than ADP-ACS or acetate kinase. Cell suspensions of Hc. saccharolyticus metabolized acetate only when they contained AMP-ACS activity, i.e., when they were obtained after growth on acetate or from the stationary phase after growth on glucose. Suspensions of exponential glucose-grown cells, containing only ADP-ACS but not AMP-ACS, did not consume acetate. Similar results were obtained for the phylogenetic distantly related halophilic archaea Hf. volcanii and Hf. saccharovorum. We conclude that, in halophilic archaea, the formation of acetate from acetyl-CoA is catalyzed by ADP-ACS, whereas the activation of acetate to acetyl-CoA is mediated by an inducible AMP-ACS.Abbreviations. Hc. Halococcus - Hf. Haloferax - Hr. Halorubrum - Hb. Halobacterium An erratum to this article can be found at     

Origins of blood acetate in the rat.

1. Fluorimetric techniques were used to characterize the environment of tryptophan residues in thermolysin and apo-thermolysin. The apo-thermolysin was obtained by dissolving the enzyme in the presence of 10mm-EDTA, which removed the functional Zn(2+) ion and the four Ca(2+) ions/molecule from the enzyme. 2. At 25 degrees C in aqueous solution the fluorescence-emission spectrum of the native holoenzyme, on excitation at 290nm, was essentially characteristic of tryptophan, with an emission maximum at 333nm. The emission maximum of the apoenzyme is red-shifted to 338nm and the relative intensity of fluorescence is decreased by 10%, both effects indicating some unfolding of the protein molecule, with the indole groups being transferred to a more hydrophilic environment. 3. Fluorescence quenching studies using KI, N'-methylnicotinamide hydrochloride and acrylamide indicated a more open structure in the apoenzyme, with the tryptophan residues located in a negatively charged environment. 4. The thermal properties of the apoenzyme, as monitored by fluorescence-emission measurements, are dramatically changed with respect to the native holoenzyme. In fact, whereas the native enzyme is heat-stable up to about 80 degrees C, for the apoenzyme a thermal transition is observed near 48 degrees C. The apoenzyme is also unstable to the action of unfolding agents such as urea and guanidinium chloride, much as for other globular proteins from mesophilic organisms. 5. The functional Zn(2+) ion does not contribute noticeably to the stability of thermolysin. 6. It is concluded that a major role in the structural stability of thermolysin is played by the Ca(2+) ions, which have a bridging function within this disulphide-free protein molecule.     

Ethyl acetate production in the gas phase

Summary Ethyl acetate was produced from gaseous ethanol and acetic acid using a lipase from Porcine Pancreas at 25 °C. Its production was investigated at different moisture content of lipase enzyme powder because it is known that moisture content of enzyme affects the reaction in the gas phase.     

Effects of cyproterone acetate in the male Japanese quail

Male Japanese Quail were injected with oil or with one of four dosages of cyproterone acetate for 8 days. Copulatory behavior and cloacal gland size were measured daily. Cyproterone acetate reduced cloacal gland size at all dosages and in two experiments reduced copulation in eight out of 17 males at the two highest dosages. Thus, cyproterone acetate results in reproductive changes in at least two classes of vertebrates, and has behavioral as well as morphological effects. The morphological effects are more striking, however.     

Kinetics of the methanogenic fermentation of acetate

Inhibition of the fermentation of acetate to methane and carbon dioxide by acetate was analyzed with an acetate-acclimatized sludge and with Methanosarcina barkeri Fusaro under mesophilic conditions. A second-order substrate inhibition model, q(ch(4) ) = q(m)S/[K(s) + S + (S/K(i))], where S was the concentration of undissociated acetic acid, not ionized acetic acid, could be applicable in both cases. The analysis resulted in substrate saturation constants, K(s), of 4.0 muM for the acclimatized sludge and 104 muM for M. barkeri. The threshold concentrations of undissociated acetic acid when no further acetate utilization was observed were 0.078 muM (pH 7.50) for the acclimatized sludge and 4.43 muM (pH 7.45) for M. barkeri. These kinetic results suggested that the concentration of undissociated acetic acid became a key factor governing the actual threshold acetate concentration for acetate utilization and that the acclimatized sludge in which Methanothrix spp. appeared dominant could utilize acetate better and survive at a lower concentration of undissociated acetic acid than could M. barkeri.     

The mechanism of acetate assimilation in purple nonsulfur bacteria lacking the glyoxylate pathway: acetate assimilation in Rhodobacter sphaeroides cells

The mechanism of acetate assimilation in the purple nonsulfur bacterium Rhodobacter sphaeroides, which lacks the glyoxylate pathway, is studied. It is found that the growth of this bacterium in batch and continuous cultures and the assimilation of acetate in cell suspensions are not stimulated by bicarbonate. The consumption of acetate is accompanied by the excretion of glyoxylate and pyruvate into the medium, stimulated by glyoxylate and pyruvate, and inhibited by citramalate. The respiration of cells in the presence of acetate is stimulated by glyoxylate, pyruvate, citramalate, and mesaconate. These data suggest that the citramalate cycle may function in Rba. sphaeroides in the form of an anaplerotic pathway instead of the glyoxylate pathway. At the same time, the low ratio of fixation rates for bicarbonate and acetate exhibited by the Rba. sphaeroides cells (approximately 0.1), as well as the absence of the stimulatory effect of acetate on the fixation of bicarbonate in the presence of the Calvin cycle inhibitor iodoacetate, suggests that pyruvate synthase is not involved in acetate assimilation in the bacterium Rba. sphaeroides.     

Transport of acetate and butyrate in the hind-gut of rabbits

1. Everted sacs of colonic mucosa from the wild rabbit did not transport acetate against a concentration gradient, but permeation down a concentration gradient did occur. 2. Butyrate was shown to permeate sheets of caecal mucosa with some conversion into ketone bodies during the passage. More ketone bodies were released from the serosal surface of the sheet than from the epithelial surface, regardless of the side to which the butyrate was added. 3. During absorption in vivo of [1-(14)C]butyrate from the caecum the ratio of [(14)C]butyrate to (14)C-labelled ketone bodies in the blood collected from the appropriate caecal vein was 13. The extent of conversion of butyrate into ketone bodies during absorption in vivo was less than that observed during transport in vitro. Possible explanations of these differences are discussed. 4. The relative concentrations of the individual volatile fatty acids in blood collected from the caecal vein during absorption in vivo were similar to those present in contents from the caecum. 5. The results are compared with similar transport and absorption studies on the ruminant fore-stomach.     

Medroxyprogesterone acetate in human serum

Conjugates of medroxyprogesterone acetate (MPA) in human serum are investigated using chromatography and techniques (equilibrium dialysis, gel filtration, and polyacrylamide gel electrophoresis) previously described for studying the binding of MPA. 17 serum samples were obtained from 7 women at various times after the intramuscular injection of 150 mg Depo-Provera. Mean concentration of MPA in the unconjugated fraction of serum was 3.9 mg/ml (range 0.8-10.7 ng/ml); in the conjugated fraction, the value was 2.7 ng/ml (range 0.6-11.4 ng/ml), a mean value of 81.7% (range 18.4-286%) of that in the unconjugated fraction. The conjugate appears to be mainly a glucuronide since solvolysis released only small amounts of MPA. MPA metabolites were also detected in blood. The MPA levels in blood measured by radioimmunoassay were generally lower when serum was extracted with an organic solvent rather than when the assay was carried out directly in the serum. This finding suggests the presence in blood of either MPA in a conjugated form or metabolites interacting with the antiserum which were not extracted by the solvents used. Equilibrium dialysis showed that undiluted plasma bound 85.8% of triated hydrogen-MPA; with increasing dilution of the plasma, the amount of bound triated hydrogen-MPA decreased. The apparent association constant calculated according to the method of Vermeulen and Verdonck was 2.6 x 10 4 1/mol. MPA appeared to be loosely bound to albumin in blood but there was no specific binding protein for the steroid. MPA conversion to the glucuronide may be 1 of the factors regulating the level of the unconjugated but presumably biologically active steroid in blood.