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A Goldman

Publications and source records attributed to A Goldman.

At least 91 records · Page 5Linked to original sources
PubMed1996

Structure of Escherichia coli inorganic pyrophosphatase at 2.2 A resolution.

The refined crystal structures of hexameric soluble inorganic pyrophosphatase from E. coli (E-PPase) are reported to R factors of 18.7 and 18.3% at 2.15 and 2.2 A, respectively. The first contains one independent monomer; the other, two independent monomers, in an R32 unit cell. Because the E-PPase monomer is small with a large open active site, there are relatively few hydrophobic interactions that connect the active-site loops to the five-stranded twisted beta-barrel that is the hydrophobic core of the molecule. The active-site loops are, however, held in place by interactions between monomers around the threefold and twofold symmetry axes of the D(3) hexamer. Consequently, mutations of active-site residues (such as Glu20 and Lysl04) often affect protein stability and oligomeric structure. Conversely, mutations of residues in the interface between monomers (such as His136 and Hisl40) not only affect oligomeric structure but also affect active-site function. The effects of the H136Q and H140Q variants can be explained by the extended ionic interaction between H140, D143 and H136' of the neighbouring monomer. This interaction is further buttressed by an extensive hydrogen-bonding network that appears to explain why the E-PPase hexamer is so stable and also why the H136Q and H140Q variant proteins are less stable as hexamers.

Journal Article↗
PubMed1996

Catalysis by Escherichia coli inorganic pyrophosphatase: pH and Mg2+ dependence.

Steady-state rates of PPi hydrolysis by Escherichia coli inorganic pyrophosphatase (E-PPase) were measured as a function of magnesium pyrophosphatase (substrate) and free Mg2+ ion (activator) in the pH range 6.0-10.0. Computer fitting of hydrolysis data in combination with direct measures of Mg2+ binding to enzyme has resulted in a model that quantitatively accounts for our results. The major features of this model are the following: (a) E-PPase catalysis proceeds both with three and with four (and possibly with five) Mg2+ ions per active site; (b) catalysis requires both an essential base and an essential acid, and the pKas of these groups are modulated by the stoichiometry of bound Mg2+; and (c) the four-metal route predominates for concentrations of free Mg2+>0.2mM. The model straightforwardly accounts for the apparent linkage between increased pKa of an essential base and activity requirements for higher Mg2+ concentration observed for several active site variants. Microscopic rate constants for overall catalysis of PPi-Pi equilibration were determined at pH 6.5-9.3 by combined analysis of enzyme-bound PPi formation and rates of PPi hydrolysis, PPi synthesis, and Pi-H2O oxygen exchange. The catalytic activity of E-PPase at saturating substrate increases toward PPi hydrolysis and decreases toward PPi synthesis and Pi-H2O oxygen exchange with increasing pH. These changes are mainly due to an increased rate of dissociation of the second released Pi and a decreased rate of enzyme-bound PPi synthesis from enzyme-bound Pi, respectively, as the pH is raised .

Catalysis↗
PubMed1996

Effect of E20D substitution in the active site of Escherichia coli inorganic pyrophosphatase on its quaternary structure and catalytic properties.

Glutamic acid 20 is an evolutionarily conserved residue found within the active site of the inorganic pyrophosphatase of Escherichia coli (E-PPase). Here we determine the effect of E20D substitution on the quaternary structure and catalytic properties of E-PPase. In contrast to wild-type enzyme, which is hexameric under a variety of conditions, E20D-PPase can be dissociated by dilution into nearly inactive trimers, as shown by electrophoresis of cross-linked enzyme, analytical ultracentrifugation, and measurement of catalytic activity as a function of enzyme concentration. Hexamer stability is increased in the presence of both substrate and Mg2+, is maximal at pH 6.5, and falls off sharply as the pH is lowered or raised from this value. Measured at saturating substrate, 20 mM Mg2+ and pH 7.2, E20D substitution (a) decreases activity towards inorganic pyrophosphate (PPi) hydrolysis and oxygen exchange between water and inorganic phosphate (P1), (b) increases the rate of net PPi synthesis, and (c) decreases the amount of enzyme-bound PPi in equilibrium with Pi in solution. Measurements of PPi hydrolysis rate as a function of both Mg2+ concentration and pH for the E20D variant show that its decreased activity is largely accounted for on the basis of an increased pKa of the catalytically essential base at the active site, and the need for a Mg2+ stoichiometry of 5 in the enzyme-substrate complex, similar to what is seen for the D97E variant. By contrast, wild-type PPase catalysis over a wide range of Mg2+ concentration and pH is dominated by an enzyme-substrate complex having a total of four Mg2+ ions. These results are consistent with a supporting role for Glu20 in PPase catalysis and demostrate that even conservative mutation at the active site can perturb the quaternary structure of the enzyme.

Binding Sites↗
PubMed1996

Crystallographic identification of metal-binding sites in Escherichia coli inorganic pyrophosphatase.

We report refined crystal structures of the hexameric soluble inorganic pyrophosphatase from Escherichia coli (E-PPase) to R-factors of 18.3% and 17.1% at 2.2 and 2.3 angstroms, respectively. Both structures contain two independent monomers in the asymmetric unit of an R32 cell. The difference between the structures is that the latter contains 1.5 Mg2+ ions per monomer. One metal ion binds to the "tight" metal-binding site identified by equilibrium dialysis studies, and is coordinated to Asp65, Asp70, and Asp102. The other metal ion, shared between two monomers at a hitherto unidentified metal-binding site in the dyad interface between trimers, is coordinated through water molecules to Asp26s and Asn24s from two monomers. The hexamers with metal bound to them are more tightly associated than the ones without metal bound to them. Combined with our other mechanistic and structural data, the results suggest that, at high metal concentrations, E-PPase may bind at least 4.5 metals per monomer: two in the active site before binding substrate, two with substrate, and 0.5 in the dyad interface. Glu20 interacts via a water molecule with Asp70 and appears in the related yeast PPase structure (Heikinheimo, manuscript in preparation) to be involved in binding the second metal ion. Magnesium ion therefore stabilizes the hexamer form through both direct and indirect effects. The direct effect is by tighter association at the subunit interface; the indirect effect occurs because magnesium stabilizes the correct conformation of the loop between Glu20 and Ile32, a loop involved a trimer-trimmer interactions. Our results thus provide a structural explanation for the solution studies that show that the E20D variant is partially hexameric and that the hexamer form can be stabilized by binding magnesium ion.

Amino Acids↗
PubMed1996

Crystallization and preliminary crystallographic analysis of 3-carboxy-cis,cis-muconate lactonizing enzyme from Neurospora crassa.

Crystals of 3-carboxy-cis,cis-muconate lactonizing enzyme (CMLE; E.C. 5.5.1.5) from Neurospora crassa that diffract to high resolution have been obtained. The crystals belong to the orthorhombic space group P2(1)2(1)2(1) with unit-cell dimensions a = 92.1, b = 159.7, c = 236.6 A (at 103 K) and diffract at most to 2 A resolution. The asymmetric unit of the crystals appears to contain two tetrameric CMLE molecules making up a total of 328 kDa per asymmetric unit. Both cross-linking with glutaraldehyde and cryo-cooling to 103 K have been used to facilitate data collection because the crystals are unstable in the X-ray beam; both techniques extend the crystal lifetime but cryo-cooling, unlike glutaraldehyde cross-linking, does not lower the quality of the diffraction pattern.

Journal Article↗
PubMed1996

An unusual route to thermostability disclosed by the comparison of Thermus thermophilus and Escherichia coli inorganic pyrophosphatases.

The structures of Escherichia coli soluble inorganic pyrophosphatase (E-PPase) and Thermus thermophilus soluble inorganic pyrophosphatase (T-PPase) have been compared to find the basis for the superior thermostability of T-PPase. Both enzymes are D3 hexamers and crystallize in the same space group with very similar cell dimensions. Two rather small changes occur in the T-PPase monomer: a systematic removal of Ser residues and insertion of Arg residues, but only in the C-terminal part of the protein, and more long-range ion pairs from the C-terminal helix to the rest of the molecule. Apart from the first five residues, the three-dimensional structures of E-PPase and T-PPase monomers are very similar. The one striking difference, however, is in the oligomeric interactions. In comparison with an E-PPase monomer, each T-PPase monomer is skewed by about 1 A in the xy plane, is 0.3 A closer to the center of the hexamer in the z direction, and is rotated by approximately 7 degrees about its center of gravity. Consequently, there are a number of additional hydrogen bond and ionic interactions, many of which form an interlocking network that covers all of the oligomeric surfaces. The change can also be seen in local distortions of three small loops involved in the oligomeric interfaces. The complex rigid-body motion has the effect that the hexamer is more tightly packed in T-PPase: the amount of surface area buried upon oligomerization increases by 16%. The change is sufficiently large to account for all of the increased thermostability of T-PPase over E-PPase and further supports the idea that bacterial PPases, most active as hexamers or tetramers, achieve a large measure of their stabilization through oligomerization. Rigid-body motions of entire monomers to produce tighter oligomers may be yet another way in which proteins can be made thermophilic.

Amino Acid Sequence↗
PubMed1996

Histochemical analysis of dental hard tissues following bleaching.

The effect of commonly used bleaching materials on the dental hard tissues was tested in extracted human premolars. In each tooth, the apical two-thirds of the root was removed, and the remaining tooth stump was cut longitudinally into two equal segments. The segments were cleaned, dried, and divided into six experimental groups. Each group was treated with one of the following bleaching materials: 30% hydrogen peroxide (HP), 10% carbamide peroxide (CP), sodium perborate (SP), Nu-Smile (NS), Opalescence (Op), and DentlBright (DB). Treatment consisted of immersing the specimens in the respective test material followed by incubation at 37 degrees C for 7 days. The levels of calcium, phosphorus, sulfur, and potassium were measured in the enamel, dentin, and cementum. In the enamel, a significant reduction in the calcium/phosphorus (Ca/P) ratio was found following treatment with HP. In the dentin, a significant reduction in Ca/P ratio was found following treatment with HP,CP, DB, and Op. In the cementum, a significant reduction in the Ca/P ratio was found following treatment with HP, CP, NS, and Op. Changes in sulfur and potassium levels also occurred, but were usually not statistically significant. Significant reduction in sulfur levels occurred only in the cementum following treatment with CP and SP. Sulfur levels increased significantly following treatment with NS. Significant reduction in potassium levels occurred only in the dentin following treatment with CP. It is concluded that bleaching materials may adversely affect the dental hard tissues and should be used with caution.

Borates↗
PubMed1996

A serum-mediated mechanism for concomitant resistance shared by immunogenic and non-immunogenic murine tumours.

Resistance of tumour-bearing mice to a second tumour challenge, that is concomitant resistance, was evaluated in euthymic and nude mice using nine tumours with widely different degrees of immunogenicity. Two temporally separate peaks of concomitant resistance were detected during tumour development. The first one was exhibited only by small immunogenic tumours; it was tumour specific and mediated by classical immunological T-cell-dependent mechanisms. The second peak was shared by both immunogenic and non-immunogenic large tumours; it was non-specific, thymus independent and correlated with the activity of a serum factor (neither antibody nor complement) that inhibited the in vitro proliferation of tumour cells. This factor was eluted from a Sephadex G-15 column at fractions corresponding to a molecular weight of approximately 1000 Da and it was recovered from a high-performance liquid chromatography column in one peak presenting maximum absorption at 215 and 266 nm. The data presented in this paper suggest for the first time, to our knowledge, that in spite of the differences between immunogenic and non-immunogenic tumours, a common serum-mediated mechanism seems to underlie the concomitant resistance induced by both types of tumours at late stages of tumour development.

Adenocarcinoma↗
PubMed1996

Dynamic mutation loci: allele distributions in different populations.

To assess the relative contributions of trans-acting factors (replication and repair functions) and cis-acting elements (repeat and flanking DNA composition) to the mechanism of trinucleotide repeat sequence mutation we have analysed the distribution of copy number polymorphisms at 12 loci associated with dynamic mutations in 15 populations of different ethnic origins. Genome wide instability of repeats in a particular population would be evidence of trans-acting factor instigation of the mutation process, whereas instability at a particular locus (perhaps even in several populations) would be evidence that the composition of the particular locus was the most significant factor contributing to mutation. The FRA16A locus is highly polymorphic in only the European population. Some other loci exhibit distinct distributions of alleles between different populations. Therefore sequences in the vicinity of the repeat -- the cis component of a particular locus -- appear(s) to be more important in the mutation mechanism than sporadic genome-wide instability induced by trans-acting factors such as the DNA mismatch repair enzymes.

Alleles↗
PubMed1996

Ethnicity and myotonic dystrophy: a possible explanation for its absence in sub-Saharan Africa.

The CTG trinucleotide repeat, in the myotonic dystrophy (DM) myotonin protein kinase gene, was studied by PCR analysis in a total of 246 unrelated South African Bantu-speaking Negroids, 116 San and 27 Pygmies. The size and distribution of the CTG repeat were determined and showed that the alleles ranged in length from 5 to 22 repeats. The most common CTG repeat is 5 (25% of chromosomes) in the South African Negroids but 11 (27% of chromosomes) in the San population and 12 (22% of chromosomes) in the Pygmies. The southern African Bantu-speaking Negroids and San were found to have significantly fewer large repeat length alleles than do Caucasoid and Japanese populations. Since DM has not been observed in southern African Negroids, it is possible that the occurrence of fewer large CTG repeats in the normal range may, in part, explain this absence. It seems likely, that the rare DM mutation event postulated to have occurred on a specific chromosomal haplotype, occurred after the migration of humans from Africa.

Africa South of the Sahara↗
PubMed1996

Surface morphology changes in human enamel, dentin and cementum following bleaching: a scanning electron microscopy study.

Extracted human premolars were cut, cleaned and divided into 6 experimental groups. Each group was treated with one of the following bleaching materials: 30% hydrogen peroxide, 10% carbamide peroxide, sodium perborate, Nu-Smile, Opalescence and DentlBright. Morphological changes in tooth surface occurred following treatment with most bleaching agents. Hydrogen peroxide and DentlBright were associated with surface changes in all dental tissues. Hydrogen peroxide, DentlBright, Nu-Smile and Opalescence were mainly associated with surface changes in the cementum, which exhibited more changes than the other tissues.

Borates↗
PubMed1996

First report of CFTR mutations in black cystic fibrosis patients of southern African origin.

Cystic fibrosis (CF) is thought to be rare in the black populations of Africa who have minimal white admixture. Only a few cases have been reported but have not been studied at the molecular level. We report the detection of CFTR mutations in three southern African black patients. One was homozygous for the 3120 + 1G-->A mutation, while the other two were compound heterozygotes each with this mutation on one chromosome. The other mutations were G1249E and a previously unreported in frame 54 bp deletion within exon 17a involving nucleotides 3196-3249 (3196del54). The 3120 + 1G-->A mutation was first described in American black patients and has been shown to be a common mutation in this population (9-14% of CF chromosomes). It was also found in a black CF patient whose father, the 3120 + 1G-->A carrier, is from Cameroon. These data suggest that it is an old mutation which accounts for many of the CFTR mutations in African blacks.

Adult↗
PubMed1996

Beta-globin haplotype analysis suggests that a major source of Malagasy ancestry is derived from Bantu-speaking Negroids.

The origins of the inhabitants of Madagascar have not been fully resolved. Anthropological studies and preliminary genetic data point to two main sources of ancestry of the Malagasy, namely, Indonesian and African, with additional contributions from India and Arabia. The sickle-cell (beta s) mutation is found in populations of African and Indian origin. The frequency of the beta s-globin gene, derived from 1,425 Malagasy individuals, varies from 0 in some highland populations to .25 in some coastal populations. The beta s mutation is thought to have arisen at least five times, on the basis of the presence of five distinct beta s-associated haplotypes, each found in a separate geographic area. Twenty-five of the 35 Malagasy beta s haplotypes were of the typical "Bantu" type, 1 "Senegal" haplotype was found, and 2 rare or atypical haplotypes were observed; the remaining 7 haplotypes were consistent with the Bantu haplotype. The Bantu beta s mutation is thought to have been introduced into Madagascar by Bantu-speaking immigrants (colonists or slaves) from central or east Africa. The Senegal beta s mutation may have been introduced to the island via Portuguese naval explorers. This study provides the first definitive biological evidence that a major component of Malagasy ancestry is derived from African populations, in particular, Bantu-speaking Negroids. beta A haplotypes are also consistent with the claim for a significant African contribution to Malagasy ancestry but are also suggestive of Asian/Oceanic and Caucasoid admixture within the Malagasy population.

Africa↗
PubMed1996

Founder effect and prevalence of myotonic dystrophy in South Africans: molecular studies.

A high prevalence of myotonic dystrophy (DM) has been described in South African Caucasoid Afrikaans-speaking families in the northern Transvaal. Evidence is presented for a strong founder effect, with a single haplotype occurring on 68% of all Caucasoid DM chromosomes; among the Afrikaans speakers, the proportion was 83%. In addition to this major haplotype, five minor DM haplotypes in the Caucasoids and two minor haplotypes in DM individuals of mixed ancestry were found. All DM chromosomes, however, had a common haplotype core, namely, Alu (ins), HinfI-2 (intron 9), and TaqI-2 (D19S463). We have detected significant linkage disequilibrium between the DM mutation and particular alleles of the extragenic markers D19S112 and D19S207. Significant differences were found in allele and haplotype distributions in the Caucasoid DM and non-DM chromosomes and Negroid non-DM chromosomes. These findings together with the strong association of allele 3 at the D19S63 locus on 93% (14/15) of the South African DM chromosomes suggest that the majority of present-day DM mutations in South African Caucasoids may have originated from a common initial founder who introduced one of the European ancestral mutations.

Alleles↗
PubMed1995

Dissociation of hexameric Escherichia coli inorganic pyrophosphatase into trimers on His-136-->Gln or His-140-->Gln substitution and its effect on enzyme catalytic properties.

Each of the five histidines in Escherichia coli inorganic pyrophosphatase (PPase) was replaced in turn by glutamine. Significant changes in protein structure and activity were observed in the H136Q and H140Q variants only. In contrast to wild-type PPase, which is hexameric, these variants can be dissociated into trimers by dilution, as shown by analytical ultracentrifugation and cross-linking. Mg2+ and substrate stabilize the hexameric forms of both variants. The hexameric H136Q- and H140Q-PPases have the same binding affinities for magnesium ion as wild-type, but their hydrolytic activities under optimal conditions are, respectively, 225 and 110% of wild-type PPase, and their synthetic activities, 340 and 140%. The increased activity of hexameric H136Q-PPase results from an increase in the rate constants governing most of the catalytic steps in both directions. Dissociation of the hexameric H136Q and H140Q variants into trimers does not affect the catalytic constants for PPi hydrolysis between pH 6 and 9 but drastically decreases their affinities for Mg2PPi and Mg2+. These results prove that His-136 and His-140 are key residues in the dimer interface and show that hexamer formation improves the substrate binding characteristics of the active site.

Amino Acid Sequence↗
PubMed1995

The refined X-ray structure of muconate lactonizing enzyme from Pseudomonas putida PRS2000 at 1.85 A resolution.

We report here the refined X-ray crystal structure of muconate lactonizing enzyme (MLE) from Pseudomonas putida PRS2000 at a resolution of 1.85 A with an R-factor of 16.8%. An enzyme from the beta-ketoadipate pathway, MLE catalyses the conversion of cis,cis-muconate to muconolactone. It is a homo-octamer, one monomer consisting of 373 amino acid residues. MLE has two large domains and a C-terminal subdomain: an alpha + beta domain, an alpha beta-barrel domain and a C-terminal meandering subdomain. The alpha beta-barrel domain is highly irregular. Its structure is (beta/alpha)7 beta, with the structural role of the last alpha-helix being replaced by both the C-terminal subdomain and part of the N-terminal domain. The fifth, seventh and eighth barrel strands are unusual because they have left-handed twist about their axes. The strand crossing angles also vary enormously, from +9 degrees to -69 degrees; the first and last strands, which close the barrel, cross at an angle of -69 degrees, making extensive strand-strand hydrogen bonding impossible. The first barrel strand is also unusual because it starts in the N-terminal domain and forms hydrogen bonds to the C-terminal subdomain beta-sheet as well as to its neighbouring strands in the barrel. It thus cements the whole protein together. As in other alpha beta-barrel proteins, the active site of MLE, present in each subunit is at the C-terminal ends of the barrel beta-strands. The active site cleft contains an essential manganese ion, is lined with charged and other polar residues, and contains many of the crystallographic water molecules. The manganese ion is octahedrally co-ordinated to three side-chain carboxylate groups and three water molecules, and is at the centre of a radiating web of ionic and hydrogen-bonding interactions. Additionally, two water molecules are buried in the centre of the barrel and two hydrophilic side-chains (Lys167 and Arg196) make both hydrophobic and hydrophilic packing interactions with much of the barrel interior. The barrel interior is thus also unusual because it is so hydrophilic; the dominating force appears to be the need to solvate the metal ion effectively. This might account for the irregularity of the barrel. The catalytic mechanism has been investigated by docking both substrate and product in the active site with the C-COO- of muconolactone superimposed on the corresponding atoms of cis,cis-muconate. In agreement with earlier kinetic and spectroscopic results, the manganese ion does not interact directly with substrate or product.(ABSTRACT TRUNCATED AT 400 WORDS)

Crystallization↗
PubMed1995

How to make my blood boil.

Two recent papers comparing the structure of a hyperthermophilic protein with its mesophilic counterpart both conclude that large networks of ion-pairs are important for hyperthermostability. How and why is not yet clear.

Amino Acid Sequence↗