Biomedical subjects

J K Stoops

Publications and source records attributed to J K Stoops.

At least 37 records · Page 2Linked to original sources

PubMed1991

Structural studies of human alpha 2-macroglobulin: concordance between projected views obtained by negative-stain and cryoelectron microscopy.

Two views of native alpha 2-macroglobulin are revealed by electron microscopy of negatively stained samples; in one view the molecule resembles a padlock and in the other, a pair of lips. Interconversion of the two views upon tilting establishes that these are two different projected views of the same structure. Furthermore, the two views are related by a 45 degrees rotation about their major axis because they interconvert when the specimens are titled +/- 22.5 degrees. Negatively stained molecules on Butvar films present a nearly equal distribution of the two views, whereas in frozen-hydrated samples the molecules almost exclusively are oriented in the lip view. Measurements from both views indicate that the alpha 2-macroglobulin molecule is approximately 200 A long and approximately 140 A wide. Our results suggest that alpha 2-macroglobulin is composed of two protomeric units, each in the shape of a twisted letter S. These units are joined together at their ends to form a complex with point group symmetry 222. The 45 degrees interconversion angle between the lip and padlock views support this arrangement. Average images of unstained and stained lips are quite similar, indicating that the native structure is consistently preserved by the two electron microscopy procedures used in this investigation. This is substantiated by the interconversion between the lip and padlock views that occurs when the molecule is rotated 45 degrees [corrected] about its major twofold axis.

Cryopreservation↗

PubMed1990

The yeast fatty acid synthase. Pathway for transfer of the acetyl group from coenzyme A to the Cys-SH of the condensation site.

The reaction pathway of enzyme-catalyzed acetylation of the acyl-accepting sites of the yeast synthase, a Ser-OH at the acetyl transacylase site, a Cys-SH at the beta-ketoacyl synthase site, and the acyl carrier protein 4'-phosphopantetheine-SH (Pant-SH), has been investigated using the chromophoric substrate, p-nitrophenyl thioacetate. The stoichiometry of acetylation of the native enzyme was 3 mol of acetate bound per mol of synthase unit, alpha beta (Mr 430,000). The acetylation process is biexponential; the rate constant of acetylation of the first 2 mol is 5.0 s-1 and the third mol is 0.2 s-1. The pathway by which acetyl moiety is added to the enzyme was determined by selectively blocking the acyl-accepting sites and subsequently determining the kinetics and stoichiometry of acetylation. The dibromopropanone-treated enzyme, in which the Pant-SH and Cys-SH are alkylated, exhibited an exponential burst of approximately 1 mol/mol of synthase unit with a rate constant of 11.0 s-1. The iodoacetamide-treated enzyme, in which Cys-SH is alkylated, had a biexponential burst with a total stoichiometry of approximately 2 mol/mol of synthase unit, with rate constants of 9 and 0.2 s-1, respectively. The kinetically competent acetylation to the extent of 2 and approximately 1 mol/mol of synthase unit for both Cys-SH and Cys-SH and Pant-SH-blocked enzymes, respectively, indicated that the route of acetyl transfer in the yeast synthase is obligatorily Ser-OH----Cys-SH. The acetylation of Pant-SH (0.2 s-1) occurs with a rate insignificant to the process of fatty acid synthesis (turnover rate constant of 1.5 s-1). These conclusions are supported by experiments involving end point radiolabeling of the synthase with [1-14C]acetyl moieties using the substrate, p-nitrophenyl thio[1-14C]acetate. Native, dibromopropanone-treated, and iodoacetamide-treated enzymes bind about 3, 1, and 2 mol of acetyl/mol of synthase unit, respectively. Performic acid oxidation studies of the acetyl-labeled enzyme indicate that there is one Ser-O-acetyl formed in the native and alkylated enzymes and one Cys-S-acetyl and one Pant-S-acetyl formed in the native enzyme. Altogether, these results support our contention that the acetylation of the Pant-SH is kinetically incompetent. Thus, the yeast synthase transacetylation reactions occur by a novel process of acetyl transfer from CoA to Ser-OH----Cys-SH, which is in contrast to the transfer from CoA to Ser-OH----Pant-SH----Cys-SH catalyzed by the prokaryotic synthases.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylation↗

PubMed1990

Overexpression and mutagenesis of the catalytic domain of dihydrolipoamide acetyltransferase from Saccharomyces cerevisiae.

The inner core domain (residues approximately 221-454) of the dihydrolipoamide acetyltransferase component (E2P) of the pyruvate dehydrogenase complex from Saccharomyces cerevisiae has been overexpressed in Escherichia coli strain JM105 via the expression vector pKK233-2. The truncated E2p was purified to apparent homogeneity. It exhibited catalytic activity (acetyl transfer from [1-14C]acetyl-CoA to dihydrolipoamide) very similar to that of wild-type E2p. The appearance of the truncated and wild-type E2p was also very similar, as observed by negative-stain electron microscopy, namely, a pentagonal dodecahedron. These findings demonstrate that the active site of E2p from S. cerevisiae resides in the inner core domain, i.e., catalytic domain, and that this domain alone can undergo self-assembly. The purified truncated E2p showed a tendency to aggregate. Aggregation was prevented by genetically engineered attachment of the interdomain linker segment (residues approximately 181-220) to the catalytic domain. All dihydrolipoamide acyltransferases contain the sequence His-Xaa-Xaa-Xaa-Asp-Gly near their carboxyl termini. By analogy with chloramphenicol acetyltransferase, the highly conserved His and Asp residues were postulated to be involved in the catalytic mechanism [Guest, J. R. (1987) FEMS Microbiol. Lett. 44, 417-422]. Substitution of the sole His residue in the S. cerevisiae truncated E2p, His-427, by Asn or Ala by site-directed mutagenesis did not have a significant effect on the kcat or Km values of the truncated E2p. However, the Asp-431----Asn, Ala, or Glu substitutions resulted in a 16-, 24-, and 3.7-fold reduction, respectively, in kcat, with little change in Km values.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetyltransferases↗

PubMed1989

Electron microscope studies of human alpha 2-macroglobulin-chymotrypsin complex: demonstration that the two structures assigned to native and proteolyzed alpha 2-macroglobulin represent two views of the proteolyzed molecule.

Electron microscope studies of native and protease-bound human alpha 2-macroglobulin have led to two contradictory models for these two structures. One viewpoint maintains that the native structure has the shape of )+(, which contracts on binding of the protease to the shape of ([). An opposing view proposes that the native structure has the shape of a padlock and that )+( and ([) are the side and end views of the proteolyzed molecule. In this investigation, electron microscope studies of the alpha-chymotrypsin-treated alpha 2-macroglobulin utilizing a tilt stage have shown that the two shapes [)+( and ([)] interconvert. This demonstrates that these two shapes represent the side and end views of the proteolyzed alpha 2-macroglobulin which are related by a 90 degree rotation of the prototype molecule.

Chymotrypsin↗

PubMed1989

Complete amino acid sequence of chicken liver acyl carrier protein derived from the fatty acid synthase.

The acyl carrier protein domain of the chicken liver fatty acid synthase has been isolated after tryptic treatment of the synthase. The isolated domain functions as an acceptor of acetyl and malonyl moieties in the synthase-catalyzed transfer of these groups from their coenzyme A esters and therefore indicates that the acyl carrier protein domain exists in the complex as a discrete entity. The amino acid sequence of the acyl carrier protein was derived from analyses of peptide fragments produced by cyanogen bromide cleavage and trypsin and Staphylococcus aureus V8 protease digestions of the molecule. The isolated acyl carrier protein domain consists of 89 amino acid residues and has a calculated molecular weight of 10,127. The protein contains the phosphopantetheine group attached to the serine residue at position 38. The isolated acyl carrier protein peptide shows some sequence homology with the acyl carrier protein of Escherichia coli, particularly in the vicinity of the site of phosphopantetheine attachment, and shows extensive sequence homology with the acyl carrier protein from the uropygial gland of goose.

Acyl Carrier Protein↗

PubMed1988

Structure of native alpha 2-macroglobulin and its transformation to the protease bound form.

Well-preserved structures of native and alpha-chymotrypsin-bound alpha 2-macroglobulin were obtained by electron microscopy. Computer processing of these images has shown that the native structure has the shape of a padlock 19 nm long. It is proposed that the native alpha 2-macroglobulin consists of the juxtaposition of two protomers with one protomer shaped like a distorted letter "S" and with the other its reverse image, to form a binding site between the two protomers near the bottom of the complex. On cleavage of the subunits with chymotrypsin, the native structure condenses to 16.7 nm and rearranges so that the interaction between the protomers is near the middle. Two images of the alpha 2-macroglobulin-chymotrypsin conjugate were obtained. We suggest that these images represent the end and side view of this complex. Based on the manner in which the native structure is assembled, we propose that the proteolyzed form of alpha 2-macroglobulin is functionally asymmetric in that both protease binding sites reside on the same half of the complex.

Chymotrypsin↗

PubMed1987

Small-angle neutron-scattering and electron microscope studies of the chicken liver fatty acid synthase.

A structural model for the chicken liver fatty acid synthase is proposed based on electron microscope and small-angle neutron-scattering studies of the enzyme. The model has the overall appearance of two side by side cylinders with dimensions of 160 X 146 X 73 A, with each subunit 160 A in length and 73 A in diameter. The model was constructed by dividing each cylinder into three domains having lengths of 32, 82, and 46 A, with the domain structures in the two subunits being related to each other by a dyad axis. The model is consistent with chemical cross-linking studies which indicated that the subunits are arranged in a head to tail fashion. The cross-linking studies further showed that the beta-ketoacyl synthase active site contains a cysteine and a pantetheine residue from adjacent subunits. It is proposed that the domains which catalyze the addition of C2 units from malonate to the growing fatty acid chain lie in the crevice between the two subunits and that the two independent sets of fatty acid-synthesizing centers lie on the major axis of the model on opposite ends of the molecular dyad.

Animals↗

PubMed1985

Yeast fatty acid synthase: structure to function relationship.

The yeast fatty acid synthase is a multifunctional enzyme composed of two nonidentical subunits in an alpha 6 beta 6 complex that is active in synthesizing fatty acids. The seven catalytic activities required for fatty acid synthesis are divided between the alpha and beta subunits such that the alpha 6 beta 6 complex has six complements of each activity. It has been proposed that these are organized into six centers for fatty acid synthesis. There are different opinions regarding the operation of these centers in the alpha 6 beta 6 complex, on view being that they are functionally independent and the other proposes half-sites activity for the complex. We have attempted to distinguish between these proposals by the most direct method of active site titration, i.e., quantitation of fatty acyl product in the absence of turnover. This was accomplished by using p-nitrophenyl thioacetate and thiophenyl malonate (in place of the coenzyme A analogues) as substrates along with NADPH, thereby depriving the yeast synthase of coenzyme A required to release product as fatty acyl coenzyme A. The amount of fatty acyl product formed was quantitated by gas-liquid chromatography, as well as by direct estimation of radioactivity in the product when p-nitrophenyl thio [1-14C] acetate was used as a substrate. In both cases, a stoichiometry of close to six was found for mole of fatty acid synthesized per mole of alpha 6 beta 6 complex. This indicates that there are six functional centers for fatty acid synthesis in the multifunctional yeast alpha 6 beta 6 fatty acid synthase and that these centers operate independently.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbon Radioisotopes↗

PubMed1985

The development and application of a novel chromophoric substrate for investigation of the mechanism of yeast fatty acid synthase.

The acetyl transacylase activity of the fatty acid synthase from yeast has been investigated using p-nitrophenylthiol acetate. The chromophoric nature of the nitrophenylthiol moiety affords a convenient spectrophotometric assay for the transacylase function as well as a means to investigate the kinetics and the mechanism of this process. A probable kinetic scheme for enzyme catalyzed transacetylation from p-nitrophenylthiol acetate to an acyl acceptor (CoA or N-acetylcysteamine) is proposed and the kinetic constants for acetylation of enzyme and for acetyl transfer to an acceptor were determined. It was also demonstrated that p-nitrophenylthiol acetate can replace acetyl-CoA as a substrate in fatty acid synthesis.

Acetyl Coenzyme A↗

PubMed1984

On the question of half- or full-site reactivity of animal fatty acid synthetase.

Our model of the animal fatty acid synthetase describes a head-to-tail arrangement of two identical subunits and predicts the presence of two centers for fatty acid synthesis. Current experiments which support this conclusion were conducted using the following approach. The thioesterase component of chicken liver fatty acid synthetase was either inhibited using phenylmethanesulfonyl fluoride or diisopropyl fluorophosphate, or released from the synthetase by limited proteolysis with alpha-chymotrypsin, thus ensuring that the fatty acyl products remain bound to the enzyme. Employing such preparations, the amount of NADPH oxidized in the initial burst of fatty acid synthesis was determined by stopped flow techniques. Gas-liquid chromatography showed that C20:0 and C22:0 constituted 85% of the fatty acids formed de novo, a result that was confirmed using [14C]acetyl-CoA in the reaction. These data showed that 1.0 mol of fatty acyl-enzyme product was formed per mol of phosphopantetheine; in addition, the measured stoichiometry of NADPH oxidation was sufficient to account for de novo fatty acid synthesis. Altogether, these results indicate that the two sites for fatty acid synthesis are active and function simultaneously. They also indicate that the thioesterase plays a key role in determining the chain specificity of fatty acid synthesis.

Acetyl Coenzyme A↗

PubMed1983

The arrangement and role of some of the amino acid residues in the beta-ketoacyl synthetase site of chicken liver fatty acid synthetase.

The beta-ketoacyl synthetase site of eukaryotic fatty acid synthetases is comprised in part of a pantetheinyl residue on one subunit juxtapositioned with a cysteinyl residue on the adjacent subunit. The present study has confirmed this arrangement and has identified 2 additional residues in the site. The active site residues were identified as summarized below. Sodium borohydride reduction of the keto derivatives of the dibromopropanone cross-linked residues yielded the alcohol derivatives which were amenable to isolation in good yields. The active enzyme yielded primarily a cysteinecysteamine derivative of 2-propanol, demonstrating that a cystyl and the pantetheinyl residues were cross-linked by dibromopropanone. However, in the cold-inactivated enzyme, the primary product of the cross-linking reaction was the dicystyl derivative. In addition, cross-linking between the cystyl and pantetheinyl residues, but not the two cystyl residues, resulted in the cross-linking of the two subunits. Therefore, it is proposed that there are two cystyl residues on one subunit juxtapositioned with the pantetheinyl residue on the adjacent subunit. The cystyl residues are highly reactive toward alkylating agents at pH 6.5, suggesting the presence of a cationic residue interacting with the thiolate anion. This proposal was supported using the bifunctional reagent o-phthalaldehyde which was found to cross-link the epsilon-amino group of lysine with the pantetheinyl-SH or the cystyl-SH in the beta-ketoacyl synthetase site to form a thioisoindole ring. The dialdehyde inhibited the enzyme by inactivating the beta-ketoacyl synthetase activity, and the inhibition could be prevented by malonyl-CoA and to a lesser extent by acetyl-CoA. Blocking the reactive thiol groups with dibromopropanone or 5,5'-dithiobis(2-nitrobenzoic acid) reduced the formation of the fluorescent thioisoindole ring. The close arrangement of a cystyl-SH, the pantetheinyl-SH, and the epsilon-amino group of lysine led us to propose that the positive epsilon-amino group may serve as an electron sink in a general acid-catalyzed decarboxylation reaction.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗

PubMed1983

Inactivation of yeast fatty acid synthetase by modifying the beta-ketoacyl reductase active lysine residue with pyridoxal 5'-phosphate.

Treatment of yeast fatty acid synthetase with pyridoxal 5'-phosphate inhibited the enzyme. Assays of the partial activities of the pyridoxal phosphate-treated synthetase showed that only the beta-ketoacyl reductase was significantly inhibited. NADPH prevented inactivation of the enzyme by pyridoxal phosphate, indicating that pyridoxal modifies a residue near or in the beta-ketoacyl reductase site. The pyridoxal-treated synthetase shows a fluorescence spectrum with a maximum of 426 nm after uv irradiation at 325 nm. Binding of the pyridoxal phosphate to the synthetase is reversible as shown by the disappearance of the fluorescence band after dialysis of pyridoxal-treated enzyme. Reduction with NaBH4 of the pyridoxal-treated enzyme eliminates this fluorescence maximum and causes the appearance of a new band at 393 nm. These observations suggest that pyridoxal phosphate interacts with the synthetase by forming a Schiff base with lysine residue at the beta-ketoacyl reductase site. Amino acid analyses of the HCl hydrolysates of the borohydride-reduced, pyridoxal-treated synthetase showed the presence of 6 mol of N6-pyridoxal derivative of lysine per mole of fatty acid synthetase, indicating the presence of six sites of beta-ketoacyl reductase in the native enzyme. Autoradiography of sodium dodecyl sulfate-polyacrylamide gels of the pyridoxal phosphate enzyme reduced with NaB3H4 indicates that the alpha subunit contains the beta-ketoacyl reductase domain. These findings are consistent with the proposed structure of the alpha 6 beta 6 complex required for palmitoyl-CoA synthesis.

Alcohol Oxidoreductases↗

PubMed1983