Search PubMed⌕ Search

Biomedical subjects

A Goldman

Publications and source records attributed to A Goldman.

At least 19 recordsLinked to original sources
PubMed1999

Pyruvate formate lyase is structurally homologous to type I ribonucleotide reductase.

BACKGROUND: Pyruvate formate lyase (PFL) catalyses a key step in Escherichia coli anaerobic glycolysis by converting pyruvate and CoA to formate and acetylCoA. The PFL mechanism involves an unusual radical cleavage of pyruvate, involving an essential C alpha radical of Gly734 and two cysteine residues, Cys418 and Cys419, which may form thiyl radicals required for catalysis. We undertook this study to understand the structural basis for catalysis. RESULTS: The first structure of a fragment of PFL (residues 1-624) at 2.8 A resolution shows an unusual barrel-like structure, with a catalytic beta finger carrying Cys418 and Cys419 inserted into the centre of the barrel. Several residues near the active-site cysteines can be ascribed roles in the catalytic mechanism: Arg176 and Arg435 are positioned near Cys419 and may bind pyruvate/formate and Trp333 partially buries Cys418. Both cysteine residues are accessible to each other owing to their cis relationship at the tip of the beta finger. Finally, two clefts that may serve as binding sites for CoA and pyruvate have been identified. CONCLUSIONS: PFL has striking structural homology to the aerobic ribonucleotide reductase (RNR): the superposition of PFL and RNR includes eight of the ten strands in the unusual RNR alpha/beta barrel as well as the beta finger, which carries key catalytic residues in both enzymes. This provides the first structural proof that RNRs and PFLs are related by divergent evolution from a common ancestor.

Acetyltransferases↗
PubMed1999

Evolutionary aspects of inorganic pyrophosphatase.

Based on the primary structure, soluble inorganic pyrophosphatases can be divided into two families which exhibit no sequence similarity to each other. Family I, comprising most of the known pyrophosphatase sequences, can be further divided into prokaryotic, plant and animal/fungal pyrophosphatases. Interestingly, plant pyrophosphatases bear a closer similarity to prokaryotic than to animal/fungal pyrophosphatases. Only 17 residues are conserved in all 37 pyrophosphatases of family I and remarkably, 15 of these residues are located at the active site. Subunit interface residues are conserved in animal/fungal but not in prokaryotic pyrophosphatases.

Amino Acid Sequence↗
PubMed1999

Of barn owls and bankers: a lush variety of alpha/beta hydrolases.

alpha/beta Hydrolase fold proteins are an important, diverse, widespread group of enzymes not yet fully exploited by structural biologists. We describe the current state of knowledge of this family, and suggest a smaller definition of the required core and some possible future avenues of exploration.

Animals↗
PubMed1999

The extreme thermostable pyrophosphatase from Sulfolobus acidocaldarius: enzymatic and comparative biophysical characterization.

Recombinant pyrophosphatase from the hyperthermophilic archaebacterium Sulfolobus acidocaldarius (S-PPase) has been heterologously expressed in Escherichia coli and could be purified in large quantities. S-PPase, previously described as a tetrameric enzyme, was shown to be a homohexameric protein that had catalytic activity with Mg2+ > Zn2+ > Co2+ >> Mn2+ >> Ni2+, Ca2+. CD and FTIR spectra demonstrate a similar overall fold for S-PPase and PPases from E. coli (E-PPase) and Thermus thermophilus (T-PPase). The relative proportions of secondary structure elements in S-PPase are close to those of a previously proposed model. S-PPase is extremely heat resistant. Even at 95 degrees C the half-life of catalytic activity is 2.5 h, which is dramatically increased in the presence of divalent cations. More than one Mg2+ per monomer is needed for catalysis, but no more than one Mg2+ per monomer is sufficient for thermal stabilization. The Tm values for S-PPase are 89 degrees C (+EDTA), 99 degrees C (+Mg2+), and >100 degrees C (+Mn2+), compared to 58 degrees C (+EDTA), 84 degrees C (+Mg2+), and 93 degrees C (+Mn2+) for E-PPase and 86 degrees C (+EDTA), 99 degrees C (+Mg2+), and 96 degrees C (+Mn2+) for T-PPase. The guanidium hydrochloride-induced unfolding follows an unknown mechanism with a biphasic kinetic and an unstable intermediate. Unfolding curves of the S-, E-, and T-PPase are independent of the method applied (CD spectroscopy and fluorescence) and show a sigmoidal and monophasic transition, indicating a change in global structure during unfolding, which can be described by a two-state process comprising dissociation and denaturation of the folded hexamer into six monomers. The respective DeltaGN-->D(25 degrees C) values of the three PPases vary from 220 to 290 kJ/mol for the overall process and are not significantly higher for the two thermophilic PPases. The stabilizing effect of Mg2+ DeltaDeltaG(25 degrees C) is 16 kJ/mol for E-PPase and 5.5-8 kJ/mol for S-PPase and T-PPase.

Bacterial Proteins↗
PubMed1999

Directed mutagenesis studies of the metal binding site at the subunit interface of Escherichia coli inorganic pyrophosphatase.

Recent crystallographic studies on Escherichia coli inorganic pyrophosphatase (E-PPase) have identified three Mg2+ ions/enzyme hexamer in water-filled cavities formed by Asn24, Ala25, and Asp26 at the trimer-trimer interface (Kankare, J., Salminen, T., Lahti, R., Cooperman, B., Baykov, A. A., and Goldman, A. (1996) Biochemistry 35, 4670-4677). Here we show that D26S and D26N substitutions decrease the stoichiometry of tight Mg2+ binding to E-PPase by approximately 0.5 mol/mol monomer and increase hexamer stability in acidic medium. Mg2+ markedly decelerates the dissociation of enzyme hexamer into trimers at pH 5.0 and accelerates hexamer formation from trimers at pH 7.2 with wild type E-PPase and the N24D variant, in contrast to the D26S and D26N variants, when little or no effect is seen. The catalytic parameters describing the dependences of enzyme activity on substrate and Mg2+ concentrations are of the same magnitude for wild type E-PPase and the three variants. The affinity of the intertrimer site for Mg2+ at pH 7.2 is intermediate between those of two Mg2+ binding sites found in the E-PPase active site. It is concluded that the metal ion binding site found at the trimer-trimer interface of E-PPase is a high affinity site whose occupancy by Mg2+ greatly stabilizes the enzyme hexamer but has little effect on catalysis.

Binding Sites↗
PubMed1999

Structural basis for the activity of two muconate cycloisomerase variants toward substituted muconates.

We have refined to 2.3 A resolution two muconate cycloisomerase (MCIase) variant structures, F329I and I54V, that differ from each other and from wild-type in their activity toward cis,cis-muconate (CCM) and substituted CCMs. The working and free R-factors for F329I are 17.4/21.6% and for I54V, 17.6/22.3% with good stereochemistry. Except for the mutated residue, there are no significant changes in structure. To understand the differences in enzymatic properties we docked substituted CCMs and CCM into the active sites of the variants and wild type. The extra space the mutations create appears to account for most of the enzymatic differences. The lack of other structural changes explains why, although structurally equivalent changes occur in chloromuconate cycloisomerase (CMCIase), the changes in themselves do not convert a MCIase into a dehalogenating CMCIase. Reanalysis of the CMCIase structure revealed only one general acid/base, K169. The structural implication is that, in 2-chloro-CCM conversion by CMCIase, the lactone ring of 5-chloromuconolactone rotates before dehalogenation to bring the acidic C4 proton next to K169. Therefore, K169 alone performs both required protonation and deprotonation steps, the first at C5 as in MCIase, and the second, after ring rotation, at C4. This distinguishes CMCIase from alpha/beta barrel isomerases and racemases, which use two different bases.

Binding Sites↗
PubMed1999

Analysis of the nu8 + nu9 Band of HNO3, Line Positions and Intensities, and Resonances Involving the v6 = v7 = 1 Dark State.

Using a high-resolution (R = 0.0025 cm-1) Fourier transform spectrum of nitric acid recorded at room temperature in the 1100-1240 cm-1 region, it has been possible to perform a more extended analysis of the nu8 + nu9 band of HNO3 centered at 1205.7075 cm-1. As in a recent analysis of this band [W. F. Wang, P. P. Ong, T. L. Tan, E. C. Looi, and H. H. Teo, J. Mol. Spectrosc. 183, 407-413 (1997)], the Hamiltonian used for the line positions calculation takes into account, for the upper state, the DeltaK = +/-2 anharmonic resonance linking the rotational levels of the v8 = v9 = 1 "bright" vibrational state and those of the "dark" v6 = v7 = 1 vibrational state. More than 4800 lines were assigned in the nu8 + nu9 band, which involve significantly higher rotational quantum numbers than in previous works. On the other hand, and surprisingly as compared to previous studies, the nu8 + nu9 band appears to be a hybrid band. In fact, nonnegligible B-type transitions could be clearly identified among the much stronger A-type lines. Accordingly, a set of individual line intensities were measured for lines of both types and were introduced in a least-squares fit to get the A- and B-type components of the transition moment operator. Finally, a synthetic spectrum of the 8.3-µm region of HNO3 has been generated, using for the line positions and line intensities the Hamiltonian constants and the expansion of the transition moment operator which were determined in this work. In this way, the B-type and the A-type components of the nu8 + nu9 band appear to contribute for about (1/4) and (3/4), respectively, to the total band intensity. Copyright 1999 Academic Press.

Journal Article↗
PubMed1999

Population pharmacokinetics of oral morphine and its glucuronides in children receiving morphine as immediate-release liquid or sustained-release tablets for cancer pain.

OBJECTIVES: (1) To determine the pharmacokinetics of morphine, morphine-6-glucuronide (M6G), and morphine-3-glucuronide (M3G) in children with cancer receiving morphine as immediate-release morphine liquid or sustained-release tablets. (2) To determine differences with age within the group and from adults. (3) To explore relationships between plasma concentration and pain measurements. STUDY DESIGN: Blood samples were collected and plasma analyzed by high-performance liquid chromatography with electrochemical and fluorescence detection. Population pharmacokinetic parameters were derived with the program P-PHARM. RESULTS: Forty children with a median age of 11.4 years (range 1.7 to 18.7 years) received a median dose of 1.4 mg/kg/d (range 0.4 to 24.6 mg/kg/d). A median of 4 blood samples per child was collected. Plasma clearance of morphine was 23.1 mL/min per kg body weight. The volume of distribution was 5.2 L/kg. Molar ratios of M3G/morphine, M6G/morphine, and M3G/M6G were 21.1, 4.7, and 4.2, respectively. Children <11 years had significantly higher clearance and larger volume of distribution for morphine and its glucuronides than older children and adults. Regression analysis indicated average plasma morphine concentration equal to dose (mg/kg/d) x 8.6 (95% confidence interval 7.4 to 9.9). Significant pain was present in 30% of the children. Higher pain scores were recorded in children with average morphine concentrations <12 ng/mL (P <.01 MW). CONCLUSION: Age differences in morphine pharmacokinetics exist within children and compared with adults. The study supports a starting dose of 1.5 to 2. 0 mg/kg/d to provide plasma morphine concentrations >12 ng/mL in children with cancer pain unrelieved by mild to moderate strength analgesia.

Adolescent↗
PubMed1999

Characterization of two infectious mouse mammary tumour viruses: superantigenicity and tumorigenicity.

Mouse mammary tumour virus (MMTV) is a type B retrovirus that causes mammary tumours in susceptible mice. MMTV encodes a superantigen (SAg) that has the property of stimulating T-cell populations expressing a particular variable region of the T-cell receptor (TCR) beta chain (Vbeta) and needs to be presented in the context of major histocompatibility complex (MHC) class II molecules. Previously, we described two exogenous MMTV, MMTV BALB14, which encodes a superantigen that induces the deletion of Vbeta14+ Tcells, and MMTV BALB2, which encodes a SAg that induces the deletion of Vbeta2+ Tcells. We now describe their biological activity: the deletions involve both CD4+ and CD8+ populations, are progressive and can be detected in blood, lymph nodes and spleen. Such deletions reflect, at least in part, those occurring during intrathymic development. Both BALB2 and BALB14 viral variants are capable of inducing a strong increase of Vbeta-specific T cells in BALB/c mice (I-A+, I-E+). However, when injected into the footpad, their initial stimulatory capacity differs in that the presence of MHC I-E molecules is essential only for the stimulation of Vbeta2+ T cells. Both viral variants are able to induce deletion even in the absence of the I-E molecule in which case, however, deletion appears later and is less pronounced. Both exogenous MMTVs induce, at the end of a year, 30-35% of pregnancy-dependent mammary adenocarcinomas.

Adenocarcinoma↗
PubMed1999

Functional characterization of Escherichia coli inorganic pyrophosphatase in zwitterionic buffers.

Catalysis by Escherichia coli inorganic pyrophosphatase (E-PPase) was found to be strongly modulated by Tris and similar aminoalcoholic buffers used in previous studies of this enzyme. By measuring ligand-binding and catalytic properties of E-PPase in zwitterionic buffers, we found that the previous data markedly underestimate Mg(2+)-binding affinity for two of the three sites present in E-PPase (3.5- to 16-fold) and the rate constant for substrate (dimagnesium pyrophosphate) binding to monomagnesium enzyme (20- to 40-fold). By contrast, Mg(2+)-binding and substrate conversion in the enzyme-substrate complex are unaffected by buffer. These data indicate that E-PPase requires in total only three Mg2+ ions per active site for best performance, rather than four, as previously believed. As measured by equilibrium dialysis, Mg2+ binds to 2.5 sites per monomer, supporting the notion that one of the tightly binding sites is located at the trimer-trimer interface. Mg2+ binding to the subunit interface site results in increased hexamer stability with only minor consequences for catalytic activity measured in the zwitterionic buffers, whereas Mg2+ binding to this site accelerates substrate binding up to 16-fold in the presence of Tris. Structural considerations favor the notion that the aminoalcohols bind to the E-PPase active site.

Binding Sites↗
PubMed1999

Purification and crystallization of a proteolytic fragment of Escherichia coli pyruvate formate-lyase.

Under anaerobic conditions, the reaction catalysed by pyruvate formate-lyase (PFL) is the first reaction after the production of pyruvate in the glycolytic pathway. Crystallization trials with Escherichia coli PFL were unsuccessful and therefore limited proteolysis was used to produce a stable crystallizable N--terminal protein fragment by trypsin cleavage. The molecular weight of this cleavage product was found to be 69.6 kDa by MALDI MS analysis, and the DNA sequence corresponding to this fragment was cloned. The recombinant protein fragment was crystallized by sitting-drop vapour diffusion using polyethylene glycol 1000 as precipitant. The crystals, which grew to 2 mm in length and 0.2 mm in cross section, belong to the hexagonal space group P61 or P65 with cell dimensions a = b = 140.4, c = 215.3 A and two molecules per asymmetric unit. X--ray diffraction data were collected from 20 to 3.2 A resolution from a single frozen crystal on a synchrotron-radiation beamline.

Acetyltransferases↗
PubMed1999

Sulfolobus acidocaldarius inorganic pyrophosphatase: structure, thermostability, and effect of metal ion in an archael pyrophosphatase.

The first crystal structure of an inorganic pyrophosphatase (S-PPase) from an archaebacterium, the thermophile Sulfolobus acidocaldarius, has been solved by molecular replacement and refined to an R-factor of 19.7% at 2.7 A. S-PPase is a D3 homohexameric protein with one Mg2+ per active site in a position similar to, but not identical with, the first activating metal in mesophilic pyrophosphatases (PPase). In mesophilic PPases, Asp65, Asp70, and Asp102 coordinate the Mg2+, while only Asp65 and Asp102 do in S-PPase, and the Mg2+ moves by 0.7 A. S-PPase may therefore be deactivated at low temperature by mispositioning a key metal ion. The monomer S-PPase structure is very similar to that of Thermus thermophilus (T-PPase) and Escherichia coli (E-PPase), root-mean-square deviations around 1 A/Calpha. But the hexamer structures of S- and T-PPase are more tightly packed and more similar to each other than they are to that of E-PPase, as shown by the increase in surface area buried upon oligomerization. In T-PPase, Arg116 creates an interlocking ionic network to both twofold and threefold related monomers; S-PPase has hydrophilic interactions to threefold related monomers absent in both E- and T-PPase. In addition, the thermostable PPases have about 7% more hydrogen bonds per monomer than E-PPase, and, especially in S-PPase, additional ionic interactions anchor the C-terminus to the rest of the protein. Thermostability in PPases is thus due to subtle improvements in both monomer and oligomer interactions.

Amino Acid Sequence↗
PubMed1999

Cystic fibrosis carrier frequencies in populations of African origin.

Cystic fibrosis (CF) is a common autosomal recessive disorder in populations of European descent. However, very little is known about CF in populations of African origin among whom it has been believed to be extremely rare. The aim of this study was to determine if this is the case or whether it is under-reported. A CFTR mutation, 3120+1G-->A, which was first reported in three African-American CF patients, has been shown to account for 9-14% of African-American CF chromosomes. It has also been found in 4/6 CF chromosomes in South African blacks and one CF chromosome of Cameroonian origin. In order to determine the carrier frequency of the 3120+1G-->A mutation in Africa, 1360 unrelated, healthy subjects were screened. Nine carriers were identified. In addition, two out of five black CF patients with positive sweat tests were found to be heterozygous for the 3120+1G-->A mutation and two out of another four black patients with symptoms suggestive of CF, but unconfirmed by sweat tests, were heterozygous for the D1270N mutation. A further three CFTR mutations, A559T, S1255X, and 444delA, which had been found in African-American CF patients, were not identified in the patients or in over 373 healthy subjects tested. The 3120+1G-->A mutation has a carrier frequency of 1 in 91 (8/728) in South African blacks with a 95% confidence interval of 1 in 46 to 1 in 197. Since this mutation accounts for between 15% and 65% of CF chromosomes in South African blacks, a corrected CF carrier frequency would be between 1 in 14 and 1 in 59. Hence, the incidence of CF would be predicted to be between 1 in 784 and 1 in 13924 births in this population. There are several possible reasons why these people are not being detected. Some of these are misdiagnosis as chronic pulmonary infection, malnutrition, tuberculosis, infantile diarrhoea, failure to thrive, or a high infant mortality rate.

Africa↗
PubMed1998

The R78K and D117E active-site variants of Saccharomyces cerevisiae soluble inorganic pyrophosphatase: structural studies and mechanistic implications.

We have solved the structure of two active-site variants of soluble inorganic pyrophosphatases (PPase), R78K and D117K, at resolutions of 1.85 and 2.15 A and R-factors of 19.5% and 18.3%, respectively. In the R78K variant structure, the high-affinity phosphate group (P1) is missing, consistent with the wild-type structure showing a bidentate interaction between P1 and Arg78, and solution data showing a decrease in P1 affinity in the variant. The structure explains why the mutation affects P1 and pyrophosphate binding much more than would be expected by the loss of one hydrogen bond: Lys78 forms an ion-pair with Asp71, precluding an interaction with P1. The R78K variant also provides the first direct evidence that the low-affinity phosphate group (P2) can adopt the structure that we believe is the immediate product of hydrolysis, with one of the P2 oxygen atoms co-ordinated to both activating metal ions (M1 and M2). If so, the water molecule (Wat1) between M1 and M2 in wild-type PPase is, indeed, the attacking nucleophile. The D117E variant structure likewise supports our model of catalysis, as the Glu117 variant carboxylate group is positioned where Wat1 is in the wild-type: the potent Wat1 nucleophile is replaced by a carboxylate co-ordinated to two metal ions. Alternative confirmations of Glu117 may allow Wat1 to be present but at much reduced occupancy, explaining why the pKa of the nucleophile increases by three pH units, even though there is relatively little distortion of the active site. These new structures, together with parallel functional studies measuring catalytic efficiency and ligand (metal ion, PPi and Pi) binding, provide strong evidence against a proposed mechanism in which Wat1 is considered unimportant for hydrolysis. They thus support the notion that PPase shares mechanistic similarity with the "two-metal ion" mechanism of polymerases.

Amino Acid Sequence↗
PubMed1998

Analysis of failure times for multiple infections following bone marrow transplantation: an application of the multiple failure time proportional hazards model.

We report an application of the proportional hazards model for multiple failure times in a study arising from the Bone Marrow Transplant Database at the University of Minnesota. The study compared the risk of infections after transplantation for patients who received allogeneic bone marrow transplants from unrelated donors (URD) versus related donors (RD). In 249 patients there was a total of 365 infections in 2.5 years of follow-up. The multiple failure time model uses all the data and provides empirical estimates of standard errors that incorporate the within-person dependencies in the data. The estimate of relative risk associated with URD was 1.4 (naive 95 per cent confidence interval 1.14 to 1.73, empirical 1.08 to 1.79), compared to the estimate 1.6 (naive or empirical, 1.1 to 2.1) from the proportional hazards model on 165 first infections only. The multivariate model gives great flexibility in modelling, for example, in accommodating a separate base hazard function for each type of failure and in allowing analysis of intervals between infections as an alternative to analysis of time from a marker event, here transplantation.

Bone Marrow Transplantation↗
PubMed1998

Evolutionary conservation of enzymatic catalysis: quantitative comparison of the effects of mutation of aligned residues in Saccharomyces cerevisiae and Escherichia coli inorganic pyrophosphatases on enzymatic activity.

Soluble inorganic pyrophosphatase (PPase) is one of the better understood phosphoryl-transfer enzymes and is distinctive in having four divalent metal ions at the active site. Here we determine pH profiles for wild-type Saccharomyces cerevisiae PPase (Y-PPase) and for 14 of its active site variants and consider the effects of active site mutation on the pH-independent parameters and acid dissociation constants that characterize these profiles against the framework of the proposed structure of the activated complex. The results obtained (a) support the current mechanistic model in which a hydroxide ion, stabilized by binding to two metal ions at the active site and by an extended system of hydrogen bonds within the active site, is the nucleophile that attacks enzyme-bound inorganic pyrophosphate and (b) provide evidence that the acid group that is necessary for maximal activity is a water molecule coordinated to a third metal ion, as shown by the general rise in the pKa of this group that is a consequence of almost all of the mutations. We further compare the present results to those previously observed for the corresponding mutations in Escherichia coli PPase [E-PPase; Salminen et al. (1995) Biochemistry 34, 782-791]. Such comparison provides a measure of the extent to which different portions of the active site are conserved. We find that some corresponding mutations have different effects on catalytic function, demonstrating that even in the context of very similar active sites, interactions of the mutated site with less well conserved portions of the enzyme, in this case outside the active site, can lead to different outcomes. On the other hand, one region of the active site is highly conserved, suggesting that it may represent a common feature of phosphoryl-transfer enzymes or a vestige of a primitive ur-PPase active site.

Amino Acid Sequence↗
PubMed1998

Trimeric inorganic pyrophosphatase of Escherichia coli obtained by directed mutagenesis.

Escherichia coli inorganic pyrophosphatase is a tight hexamer of identical subunits. Replacement of both His136 and His140 by Gln in the subunit interface results in an enzyme which is trimeric up to 26 mg/mL enzyme concentration in the presence of Mg2+, allowing direct measurements of Mg2+ binding to trimer by equilibrium dialysis. The results of such measurements, together with the results of activity measurements as a function of [Mg2+] and pH, indicate that Mg2+ binds more weakly to one of the three sites per monomer than it does to the equivalent site in the hexamer, suggesting this site to be located in the trimer:trimer interface. The otherwise unobtainable hexameric variant enzyme readily forms in the presence of magnesium phosphate, the product of the pyrophosphatase reaction, but rapidly dissociates on dilution into medium lacking magnesium phosphate or pyrophosphate. The kcat values are similar for the variant trimer and hexamer, but Km values are 3 orders of magnitude lower for the hexamer. Thus, while stabilizing hexamer, the two His residues, per se, are not absolutely required for active-site structure rearrangement upon hexamer formation. The reciprocal effect of hexamerization and product binding to the active site is explained by destabilization of alpha-helix A, contributing both to the active site and the subunit interface.

Enzyme Stability↗