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J Sygusch

Publications and source records attributed to J Sygusch.

At least 37 records · Page 2Linked to original sources
PubMed1990

Inactivation of mammalian fructose diphosphate aldolases by COOH terminus autophosphorylation.

Rabbit skeletal muscle and liver fructose 1,6-diphosphate aldolases autophosphorylate in the presence of inorganic phosphate at physiological and alkaline pH. ATP as well as nonhydrolyzable ATP analogues inhibits autophosphorylation. Autophosphorylation of aldolases abolishes catalytic activity, which is restored upon treatment with alkaline phosphatase. Limited proteolysis of aldolase preferentially hydrolyzes the COOH terminus and liberates a phosphorylated peptide. Treatment of rabbit aldolases with carboxypeptidase, which liberates the COOH terminal residue Tyr 363, although modifying catalytic activity does not affect autophosphorylation. Amino acid analyses are consistent with results of autophosphorylation of the COOH terminus showing residue His 361 in muscle aldolase and Tyr 361 in liver aldolase. Phosphate lability in acid pH by phosphorylated muscle aldolase but not by phosphorylated liver aldolase corroborates the amino acid assignment. Autophosphorylation of the aldolases in the crystalline state is consistent with an intramolecular mechanism. The pH dependence of autophosphorylation being dependent on the enzyme's physical state (soluble or crystalline) is not inconsistent with crystallization stabilizing a conformer having different amino acid pka values and/or reactivities than those of the soluble state.

Amino Acids↗
PubMed1990

Purification and characterization of thermostable beta-mannanase and alpha-galactosidase from Bacillus stearothermophilus.

Bacillus stearothermophilus secretes beta-mannanase and alpha-galactosidase enzymatic activities capable of hydrolyzing galactomannan substrates. Expression of the hemicellulase activities in the presence of locust bean gum was sequential, with mannanase activity preceding expression of alpha-galactosidase activity. The hemicellulase activities were purified to homogeneity by a combination of ammonium sulfate fractionation, gel filtration, hydrophobic interaction chromatography, and ion-exchange and chromatofocusing techniques. The purified beta-D-mannanase is a dimeric enzyme (162 kilodaltons) composed of subunits having identical molecular weight (73,000). Maximal activity did not vary between pH 5.5 and 7.5. The beta-D-mannanase activity exhibited thermostability, retaining nearly full activity after incubation for 24 h at 70 degrees C and pH 6.5. The enzyme displayed high specificity for galactomannan substrates, with no-secondary xylanase or cellulase activity detected. Hydrolysis of locust bean gum yielded short oligosaccharides compatible with an endo mode of substrate depolymerization. Initial rate velocities of the mannanase activity displayed substrate inhibition and yielded estimates for Vmax and Km of 455 +/- 60 U/mg and 1.5 +/- 0.3 mg/ml, respectively, at 70 degrees C and pH 6.5. The alpha-galactosidase activity corresponded to a trimeric enzyme (247 kilodaltons) having subunits of identical molecular weight (82,000). The alpha-galactosidase had maximal activity at pH 7 to 7.5 and retained full activity after 24 h of incubation at 60 degrees C. The enzyme had only limited activity on galactomannan substrates as compared with hydrolysis of p-nitrophenyl alpha-D-galactose. Kinetics of p-nitrophenyl alpha-D-galactose hydrolysis yielded linear reciprocal plots corresponding to Vmax and Km of 195 +/- 10 U/mg and 0.25 +/- 0.02 mM, respectively, at 60 degrees C and pH 7. The characterization of the mannanase activity is consistent with its potential use in enzymatic bleaching of softwood pulps.

Drug Stability↗
PubMed1990

Purification and Characterization of Three Chitosanase Activities from Bacillus megaterium P1.

Bacillus megaterium P1, a bacterial strain capable of hydrolyzing chitosan, was isolated from soil samples. Chitosan-degrading activity was induced by chitosan but not by its constituent d-glucosamine. Extracellular secretion of chitosanase reached levels corresponding to 1 U/ml under optimal conditions. Three chitosan-degrading proteins (chitosanases A, B, and C) were purified to homogeneity. Chitosanase A (43 kilodaltons) was highly specific for chitosan and represented the major chitosan-hydrolyzing species. Chitosanases B (39.5 kilodaltons) and C (22 kilodaltons) corresponded to minor activities and possessed comparable specific activities toward chitosan, chitin, and cellulose. Chitosanase A was active from pH 4.5 to 6.5 and was stable on the basis of activity up to 45 degrees C. The optimum temperature for enzymatic chitosan hydrolysis was 50 degrees C. Kinetic studies on chitosanase A suggest that the enzyme is substrate inhibited. The apparent K(m) and V(max) determined at 22 degrees C and pH 5.6 were 0.8 mg/ml and 280 U/mg, respectively. End products of chitosan hydrolysis by each of the three chitosanases were identified as glucosamine oligomers, similar to those obtained for previously reported chitosanase digestions.

Journal Article↗
PubMed1989

Recombinant anaerobic maize aldolase: overexpression, characterization, and metabolic implications.

Complementary DNA sequence of anaerobically induced cytoplasmic maize aldolase was expressed under control of the tac promoter sequence in Escherichia coli using the pKK223-3 plasmid as a vehicle. Levels of recombinant protein expressed exceeded 20 mg of soluble aldolase per liter of culture. The purified recombinant enzyme displayed the expected molecular weight and tetrameric subunit assembly on the basis of mobilities on denaturing electrophoretic gels and gel filtration, respectively. Sequencing of the NH2 terminus and amino acid composition analysis of the recombinant protein including COOH-terminal peptides agreed with the cDNA sequence. Partial kinetic characterization based on product inhibition studies was consistent with the ordered uni-bi reaction mechanism expected of aldolases. Turnover with respect to substrates Fru-1,6-P2 and Fru-1-P by the recombinant enzyme is the highest reported to date for class I aldolases. Fru-1,6-P2 cleavage rate by recombinant cytoplasmic maize enzyme is three times greater than that of the chloroplast enzyme. Fru-1-P cleavage is 8-fold greater than that of the rabbit liver isozyme and 20-fold greater than that of the rabbit muscle isozyme to which maize aldolase exhibits the greatest homology. The implications of such a high Fru-1-P turnover on carbohydrate utilization under anaerobiosis is discussed.

Amino Acid Sequence↗
PubMed1988

Sequential radiation damage in protein crystallography.

Radiation damage in protein crystals is described in terms of a sequential process of protein disordering. A new radiation-damage model has been tested against data from several protein crystals and can describe radiation damage corresponding to loss of the original intensity in excess of 80%. The model is an extension of previous models which characterize radiation damage in terms of successive conformational transitions of the protein from an undamaged to a spatially disordered to finally an amorphous state. The proposed model provides a more-general positional characterization of the disordered protein and includes, prior to the disordered state, a new dose-dependent state in which the protein conformation resembles the undamaged protein. Comparison of this model with the best previous model shows that the proposed model provides an improved fit to radiation-damage data.

Adenosine Triphosphatases↗
PubMed1987

Molecular architecture of rabbit skeletal muscle aldolase at 2.7-A resolution.

The molecular architecture of the rabbit skeletal muscle aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) tetramer has been determined to 2.7-A resolution. Solution of the three-dimensional structure of rabbit muscle aldolase utilized phase information from a single isomorphous Pt(CN)4(2-) derivative, which was combined with iterative-phase refinement based upon the noncrystallographic 222-fold symmetry exhibited by the tetramer subunits. The electron-density map calculated from the refined phases (mf = 0.72) was interpreted on the basis of the known amino acid sequence (363 amino acids per subunit). The molecular architecture of the aldolase subunit corresponds to a singly wound beta-barrel of the parallel alpha/beta class structures as has been observed in triose phosphate isomerase, pyruvate kinase, phosphogluconate aldolase, as well as others. Close contacts between tetramer subunits are virtually all between regions of hydrophobic residues. Contrary to other beta-barrel structures, the known active-site residues are located in the center of the beta-barrel and are accessible to substrate from the COOH side of the beta-barrel. Biochemical and crystallographic data suggest that the COOH-terminal region of aldolase covers the active-site pocket from the COOH side of the beta-barrel and mediates access to the active site. On the basis of sequence studies, active-site residues as well as residues lining the active-site pocket have been totally conserved throughout evolution. By comparison, homology in the COOH-terminal region is minimal. It is suggested that the amino acid sequence of the COOH-terminal region may be, in part, the basis for the variable specific activities aldolases exhibit toward their substrates.

Animals↗
PubMed1985

Structure of rabbit muscle aldolase at low resolution.

X-ray diffraction data were measured by x-ray diffractometry to 5-A resolution for both the monoclinic form of rabbit skeletal muscle aldolase (EC 4.1.2.13) and a platinum derivative. The heavy atom difference patterson was solved at 6-A resolution yielding eight distinct heavy atom sites. Choice was made of the enantiomorph and protein phases were calculated on the basis of single isomorphous replacement differences. The electron density map calculated from these phases was averaged according to the non-crystallographic molecular symmetry. Rotational symmetry analysis of native patterson and site symmetry analysis of refined heavy atom positions are consistent with the aldolase tetramer possessing a very high degree of 222 internal symmetry. The subunits in the tetramer are positioned in a tetrahedral configuration displaying a slight square planar deformation. Each subunit is roughly ellipsoidal in shape with the major axis nearly parallel to a local 2-fold axis. Prominent at the surface of each subunit were structural features resembling alpha helices. Each subunit contributes to its boundary surface at least six helices which are arranged in a barrel-like manner and possessing a right handed twist with respect to each other. Density associated with binding of substrate on the enzyme was located on the surface of each subunit. Cooperative aspects of the conformational changes produced upon substrate binding are discussed.

Animals↗
PubMed1985

Preliminary crystallographic investigation of rabbit liver aldolase.

Rabbit liver aldolase was purified by affinity elution on a CM52 ion exchanger. Crystals of rabbit liver aldolase suitable for X-ray diffraction experiments were grown from 45% saturated ammonium sulfate solution at 4 degrees C. The enzyme crystallizes in space group C222(1) having cell dimensions a = 377.02 A, b = 130.35 A, c = 80.04 A and diffracts to at least 3.5 A resolution. On the basis of a 55% solvent content there are eight aldolase tetramers in the unit cell. Rotational symmetry analysis to 6.7 A is consistent with the aldolase tetramers having a high degree of internal symmetry corresponding to point group 222. The crystallized enzyme is catalytically active.

Animals↗
PubMed1985

Phosphate ion inactivation of rabbit skeletal muscle aldolase in the crystalline state.

Catalytically active crystals of rabbit skeletal muscle aldolase are inactivated by phosphate ion and D-glyceraldehyde-3-phosphate. Four moles of phosphate are incorporated per mole of tetrameric enzyme. The inactivation rates are first order in time and demonstrate saturation behaviour. Competition inactivation experiments are consistent with the two substrates competing for the same site on the enzyme. Protection is afforded by substrates binding to the active site on the enzyme. No phosphate inactivation is observed in solution under identical experimental conditions and D-glyceraldehyde-3-phosphate inactivation in solution is unaffected by phosphate ion concentrations. Inactivation by phosphate is apparently due to an unique enzyme conformation stabilized upon protein crystallization.

Animals↗
PubMed1984

Extreme X-ray sensitive modification of type I aldolases by blue dye ligand chromatography.

Aldolases purified by Blue dye ligand chromatography from a variety of vertebrate sources crystallize at room temperature in a habit similar to the monoclinic form of rabbit skeletal muscle aldolase. Crystals of aldolases thus purified including rabbit muscle aldolase are extremely sensitive to X-ray (Cu K alpha) radiation and shatter after short exposure to X-ray radiation (less than 5 min.). Crystals of aldolases purified by other techniques possess demonstrable diffraction patterns and are stable in the X-ray beam with lifetimes of the order of days. No clear distinction could be made on the basis of different biochemical assays between aldolases purified by Blue dye chromatography and those purified by other techniques.

Animals↗
PubMed1984

Catalytic activity of rabbit skeletal muscle aldolase in the crystalline state.

The monoclinic crystalline form of aldolase from rabbit skeletal muscle grown at 29 degrees C is catalytically active in the direction of aldol cleavage. Activity was assayed for in a crystallization buffer containing 45% saturated ammonium sulfate using chemically unmodified single crystals cut to precise dimensions. Diffusion effects on velocities from assays employing aldolase crystals do not appear to be limiting when cut single crystals are crushed. Assays of crushed crystals are linear with respect to both time and enzyme concentration. Kinetic constants are reported for both substrates fructose 1-phosphate and fructose 1,6-phosphate. Maximal velocities and binding constants determined differ by no more than a factor of 2 between the crystalline and the soluble state of the enzyme. Analysis of the kinetic constants for fructose 1-phosphate as substrate shows that binding of substrate does not change in going to the crystalline state. Release of product is reduced roughly 2-fold in the crystalline state. A similar conclusion can be reached in the case of fructose 1,6-phosphate as substrate provided the "on" steps of substrate and product are only diffusion limited but independent of the physical state of the enzyme. It is not possible to distinguish between a more sluggish conformational change during catalysis or simply tighter product binding in the crystalline state as compared to the soluble enzyme state.

Animals↗
PubMed1977

Location of pyridoxal phosphate in glycogen phosphorylase a.

The pyridoxal 5'-phosphate cofactor of glycogen phosphorylase a (1,4-alpha-D-glucan:orthophosphate alpha-glucosyltransferase, EC2.4.1.1.) has been positioned on the protomer with x-ray diffraction data, chemical markers, and sequence information. The electron density was computed from 3.0-A resolution phases calculated from four heavy-atom derivatives. The cofactor is buried inside the protomer adjacent to the glucose-binding site. The phosphoryl substrates Pi and glucose-1-P each bind at two sites on the protomer. At low concentrations, Pi and glucose-1-P bind in the same location as does the allosteric effector AMP, near the monomer-monomer interface and some 30 A from the glucose site. At high concentrations glucose-1-P also binds strongly at the glucose site, with its phosphate only 7.2 A from that of the cofactor. Inorganic phosphate can also bind at this site. Implications for the participation of the pyridoxal phosphate in the catalytic mechanism are discussed in the light of these structural findings as well as the wealth of indirect evidence in the literature.

Binding Sites↗
PubMed1977

An essential arginine residue in the active-site pocket of glycogen phosporylase.

Phosphorylases a and b (EC 2.4.1.1) were inactivated by selective modification of arginyl residues on reaction with 2,3-butanedione in sodium borate buffer. The rate of inactivation was slightly greater for phosphorylase a than b. The course of inactivation followed pseudo-first-order kinetics with some deviations at low rates or at more than 60% inactivation. The rate of inactivation was first order with respect to butanedione concentration. The inactivation was partially reversible, and ultracentrifugal studies showed no change in subunit association or dissociation. Amino acid analyses indicated that several arginines were modified during inactivation and that no other amino acid was affected. Protection from inactivation was provided by the substrate glucose 1-phosphate (G1P), alone or together with the allosteric activator AMP, as well as by the competitive inhibitor UDP-glucose. The rate of inactivation of phosphorylase b was also retarded by the presence of AMP alone. Glycogen did not have any appreciable effect on inactivation. The Km of G1P for phosphorylase a remained constant over the course of inactivation, while the Km values of G1P and AMP for phosphorylase b increased. The modification of cross-linked tetragonal microcrystals of phosphorylase a followed the same trend as the enzyme in solution, although the rate of inactivation was slower. The X-ray crystallography studies at 6 A (1 A = 0.1 nm) resolution, of butanedione-treated cross-linked tetragonal crystals of phosphorylase a showed a large new peak of electron density at the end of a long side chain in the active-site pocket. The substrates G1P and arsenate, as well as UDP-glucose, had previously been shown to bind in that location. Other, small peaks of electron density were found in locations on the outside of the protein molecule. UDP-glucose failed to bind to the active site of crystals which had been treated with butanedione, while AMP, which also binds in the active-site pocket, showed a lower occupancy. This work indicates the presence of a functional arginine residue at the binding site for G1P in glycogen phosphorylases a and b.

Adenosine Monophosphate↗