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H C Watson

Publications and source records attributed to H C Watson.

At least 19 recordsLinked to original sources
PubMed1997

Major grass pollen allergen Lol p 1 binds to diesel exhaust particles: implications for asthma and air pollution.

BACKGROUND: Grass pollen allergens are known to be present in the atmosphere in a range of particle sizes from whole pollen grains (approx. 20 to 55 microns in diameter) to smaller size fractions < 2.5 microns (fine particles, PM25). These latter particles are within the respirable range and include allergen-containing starch granules released from within the grains into the atmosphere when grass pollen ruptures in rainfall and are associated with epidemics of thunderstorm asthma during the grass pollen season. The question arises whether grass pollen allergens can interact with other sources of fine particles, particularly those present during episodes of air pollution. OBJECTIVE: We propose the hypothesis that free grass pollen allergen molecules, derived from dead or burst grains and dispersed in microdroplets of water in aerosols, can bind to fine particles in polluted air. METHODS: We used diesel exhaust carbon particles (DECP) derived from the exhaust of a stationary diesel engine, natural highly purified Lol p 1, immunogold labelling with specific monoclonal antibodies and a high voltage transmission electron-microscopic imaging technique. RESULTS: DECP are visualized as small carbon spheres, each 30-60 nm in diameter, forming fractal aggregates about 1-2 microns in diameter. Here we test our hypothesis and show by in vitro experiments that the major grass pollen allergen, Lol p 1, binds to one defined class of fine particles, DECP. CONCLUSION: DECP are in the respirable size range, can bind to the major grass pollen allergen Lol p 1 under in vitro conditions and represent a possible mechanism by which allergens can become concentrated in polluted air and thus trigger attacks of asthma.

Air Pollution↗
PubMed1993

Site-directed mutagenesis of yeast phosphoglycerate kinase. Arginines 65, 121 and 168.

In the absence of a structure of the closed form of phosphoglycerate kinase we have modified by site directed mutagenesis several of the residues which, on the basis of the open form structure, are likely to be involved in substrate binding and catalysis. Here we report on the kinetic and anion activation properties of the yeast enzyme modified at positions 65, 121 and 168. In each case an arginine, thought to be involved in the binding of the sugar substrate's non-transferable phosphate group, has been replaced by lysine (same charge) and by methionine (no charge). Km values for 3-phosphoglycerate of all six mutant enzymes are only marginally higher than that of the wild-type enzyme. Removing the charge associated with two of the three arginine residues appears to influence (as judged by the measured Km's) the binding of ATP. Although binding affinity is not necessarily coupled to turnover the substitutions which have the greatest effect on the Km's do correlate with the reduction in enzymes maximum velocity. The one exception to this generalisation is the R65K mutant which, surprisingly, has a significantly higher kcat than the wild-type enzyme. In the open form structure of the pig muscle enzyme each of the three substituted arginines residues are seen to make two hydrogen bonds to the sugar substrate's non-transferable phosphate. From this it might be expected that anion activation would be similarly affected by the substitution of any one of these three residues. Although the interpretation of such effects are complicated by the fact that one of the mutants (R65M) unfolds at low salt concentrations, this appears not to be the case. Replacing Arg121 and Arg168 with methionine reduces the anion activation whereas a lysine in either of these two positions practically destroys the effect. With the substitutions at residue 65 the opposite is observed in that the lysine mutant shows anion activation whereas the methionine mutant does not.

Adenosine Triphosphate↗
PubMed1993

Structure of glycosomal glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma brucei determined from Laue data.

The three-dimensional structure of glycosomal glyceraldehyde-3-phosphate dehydrogenase [D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.12.1.12] from the sleeping-sickness parasite Trypanosoma brucei was solved by molecular replacement at 3.2-A resolution with an x-ray data set collected by the Laue method. For data collection, three crystals were exposed to the polychromatic synchrotron x-ray beam for a total of 20.5 sec. The structure was solved by using the Bacillus stearothermophilus enzyme model [Skarzyński, T., Moody, P. C. E. & Wonacott, A. J. (1987) J. Mol. Biol. 193, 171-187] with a partial data set which was 37% complete. The crystals contain six subunits per asymmetric unit, which allowed us to overcome the absence of > 60% of the reflections by 6-fold density averaging. After molecular dynamics refinement, the current molecular model has an R factor of 17.6%. Comparing the structure of the trypanosome enzyme with that of the homologous human muscle enzyme, which was determined at 2.4-A resolution, reveals important structural differences in the NAD binding region. These are of great interest for the design of specific inhibitors of the parasite enzyme.

Animals↗
PubMed1993

The structure of a thermally stable 3-phosphoglycerate kinase and a comparison with its mesophilic equivalent.

The structure of the phosphoglycerate kinase (PGK) from Bacillus stearothermophilus, a moderate thermophile, has been determined and compared with that of its mesophilic equivalent from yeast. The Bacillus enzyme structure was solved by molecular replacement and improved using constrained rigid-body, molecular dynamics and conventional refinement procedures. The refinement residual, calculated using all the measured data between 8 and 1.65 A, is 0.18(1). The stereo chemical deviations of the final model from ideality are 0.01 A for both bonds and planes. The mid-point temperatures of the Bacillus and yeast enzymes are 67 and 53 degrees C, respectively. Differential scanning calorimetry indicates that the energy difference (delta delta G) between the mesophilic and thermophilic enzymes is of the order of 5 kcal mol-1 at room temperature. The structure comparison indicates that the features most likely to be responsible for the increased thermal stability of the Bacillus enzyme are the increased internal hydrophobicity, additional ion pairs, and better alpha-helix stability resulting from the removal of helix destabilizing residues and extra helix-dipole/helix side chain ionic interactions.

Enzyme Stability↗
PubMed1993

A data-based reaction mechanism for type I fructose bisphosphate aldolase.

The structures of three type I fructose-1,6-bisphosphate aldolases have been determined and the common residues surrounding the Schiff base-forming Lys residue located. Armed with this information, it is now possible to propose a mechanism for this ubiquitous enzyme which is consistent with the recorded biochemical data. An interesting, but by no means mandatory, feature of the reaction mechanism is that catalysis can proceed without exchange with the solvent.

Animals↗
PubMed1992

Purification, crystallization and preliminary X-ray analysis of the 3-phosphoglycerate kinase from Bacillus stearothermophilus.

As part of a programme investigating the molecular basis of thermal stability in proteins we have isolated and characterized the thermally stable 3-phosphoglycerate kinase (PGK) from Bacillus stearothermophilus NCA 1503. The B. stearothermophilus PGK has been crystallized in a form suitable for X-ray diffraction analysis. Crystals which diffract to greater than 1.8 A resolution have been grown in the presence of the nucleotide substrate, MgATP, using polyethylene glycol (PEG 600) as a precipitant. The best crystals have been obtained using "seeding" techniques and are monoclinic, space group P2(1), with cell dimensions a = 40.5 A, b = 74.0 A, c = 68.5 A and beta = 99.8 degrees.

Crystallization↗
PubMed1992

Characterisation of yeast phosphoglycerate kinase modified by mutagenesis at residue 21.

Site-directed mutagenesis has been used to produce mutant forms of yeast phosphoglycerate kinase in which the conserved active-site residue, Arg21, has been replaced by a methionine or a lysine. Kinetic results obtained using these mutant enzymes show that their Km for both 3-phospho-D-glycerate and ATP are significantly different from those recorded for the wild-type enzyme. The Vmax for the lysine mutant is reduced by a factor of two from that of the wild-type enzyme whereas the Vmax for the methionine mutant is reduced more than sevenfold. A very clean electron-density-difference map shows little, if any, evidence of a structural change associated with the C-terminal domain, although resonances in the NMR spectra associated with the ATP-binding site (C-terminal domain) are also affected by the mutation as one might expect from the kinetic results. The NMR data show that binding at both the 3-phospho-D-glycerate and the non-productive ATP-binding site (associated with the N-terminal domain) are affected in the mutant in a way which is different to that associated with the wild-type enzyme. These results, taken together with the X-ray and kinetic data, indicate that the non-productive ATP-binding site and the activating anion-binding site are both associated with the basic patch region of yeast phosphoglycerate kinase.

Amino Acid Sequence↗
PubMed1992

An investigation of large inhibitors binding to phosphoglycerate kinase and their effect on anion activation.

This study extends, to a series of larger anions, our earlier investigation of the interaction of the trypanocidal drug suramin and other small negatively charged molecules with yeast phosphoglycerate kinase. 1H-NMR structural studies of phosphoglycerate kinase in the presence of varying concentrations of these large molecules (designed to mimic, at one end, the anionic charge distribution in the substrate 3-phosphoglycerate, while possibly being able to interact across the cleft of the enzyme) including inositol 1,4,5-triphosphate, 4-amino-6-trichloroethenyl-1,3- benzenedisulphonamide, gallic acid and sulphasalazine are described. The anion activation and/or inhibition of the enzyme by these molecules are also reported. Evidence that binding to the general anion site in the 'basic patch' region of the protein may be responsible for either the activating or inhibiting effects, while binding at the hydrophobic (catalytic) site leads to inhibition only is presented. A reaction scheme which explains these observations is given.

Anions↗
PubMed1991

Sequence and expression of the gene encoding 3-phosphoglycerate kinase from Bacillus stearothermophilus.

The structural gene (pgk) encoding 3-phosphoglycerate (PGK) from Bacillus stearothermophilus NCA1503, has been cloned in Escherichia coli and its complete nucleotide sequence determined. The gene consists of an open reading frame corresponding to a protein of 394 amino acids (aa) (calculated Mr 42,703) and, in common with other prokaryotic pgk genes, is preceded by the structural gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Constructs containing the B. stearothermophilus pgk gene and its flanking sequences in the high-copy plasmid, pUC9, co-express both PGK and GAPDH at high levels in transformed E. coli cells, typically producing PGK at levels of up to 30% of the soluble cell protein. The deduced aa sequence of B. stearothermophilus PGK is compared with those of the mesophilic (yeast) and extreme thermophilic (Thermus thermophilus) enzymes since the crystal structure of these PGKs are known or are in the process of being determined. Changes in the sequences of the three enzymes, as they appear to relate to the enhancement of thermal stability, are discussed.

Amino Acid Sequence↗
PubMed1991

Activity and specificity of human aldolases.

The structure of the type I fructose 1,6-bisphosphate aldolase from human muscle has been extended from 3 A to 2 A resolution. The improvement in the resulting electron density map is such that the 20 or so C-terminal residues, known to be associated with activity and isozyme specificity, have been located. The side-chain of the Schiff's base-forming lysine 229 is located towards the centre of an eight-stranded beta-barrel type structure. The C-terminal "tail" extends from the rim of the beta-barrel towards lysine 229, thus forming part of the active site of the enzyme. This structural arrangement appears to explain the difference in activity and specificity of the three tissue-specific human aldolases and helps with our understanding of the type I aldolase reaction mechanism.

Amino Acid Sequence↗
PubMed1991

A proton-NMR study of a site-directed mutation (His388----Glu) in the interdomain region of yeast phosphoglycerate kinase. Implications for domain movement.

Proton NMR has been used to study a site-directed mutant of yeast phosphoglycerate kinase in which the interdomain residue His388 has been replaced by a glutamine residue. Using 1H-NMR spectroscopy, it was found that 3-phosphoglycerate binding to the mutant protein induces different conformational effects to those observed for the wild-type enzyme. These differences are not only located at the 3-phosphoglycerate binding site but are also seen as long-range effects at the surface of the protein. Measurements of the Kd for 3-phosphoglycerate from the NMR experiments show that the mutant enzyme has a 30-times reduced affinity for this substrate as compared with the wild-type enzyme. These data are consistent with the suggestion that an aromatic residue at position 388 plays an important role in the proposed hinge-bending mechanism.

Glutamine↗
PubMed1990

The crystal structure of human muscle aldolase at 3.0 A resolution.

The three-dimensional structure of fructose-1,6-bisphosphate aldolase from human muscle has been determined at 3.0 A resolution by X-ray crystallography. The active protein is a tetramer of 4 identical subunits each of which is composed of an eight-stranded alpha/beta-barrel structure. The lysine residue responsible for Schiff base formation with the substrate is located near the centre of the barrel in the middle of the sixth beta-strand. While the overall topology of the alpha/beta-barrel is very similar to those found in several other enzymes, the distribution of charged residues inside the core of the barrel seems distinct. The quaternary fold of human muscle aldolase uses interfacial regions also involved in the subunit association of other alpha/beta-barrel proteins found in glycolysis, but exploits these regions in a manner not seen previously.

Fructose-Bisphosphate Aldolase↗
PubMed1990

Site-directed mutagenesis of aspartic acid 372 at the ATP binding site of yeast phosphoglycerate kinase: over-expression and characterization of the mutant enzyme.

A new phosphoglycerate kinase over-expression vector, pYE-PGK, has been constructed which greatly facilitates the insertion and removal of mutant enzyme genes by cleavage at newly introduced BamHI sites. This vector has been used to prepare mutant protein in appreciable (100 mg) quantities for use in kinetic, crystallographic and NMR experiments. Aspartate 372 is an invariant amino acid residue in genes known to code for a functionally active PGK. The function of this acidic residue appears to be to help desolvate the magnesium ion complexed with either ADP or ATP when this substrate binds to the enzyme. Both crystallographic and nuclear magnetic resonance experiments show that the replacement of the residue with asparagine has only minimal effects on the overall structure. The substitution of the charged carboxyl group with that of the neutral amide affects the binding of the nucleotide substrate as predicted but not, as might have been expected, the binding of 3-phosphoglycerate. The overall velocity of the enzymic reaction (Vmax) is reduced 10-fold by the substitution of aspartic acid 372 by an asparagine residue (D372N). This reduction in Vmax is considerably less than one would expect from its known position within the structure of the enzyme. This result therefore poses questions about our understanding of charged groups at the active centres of enzymes and of the reason for their apparent conservation.

Adenosine Triphosphate↗
PubMed1989

Site-directed mutagenesis of histidine 62 in the 'basic patch' region of yeast phosphoglycerate kinase.

Site-directed mutagenesis has been used to produce a mutant form of yeast phosphoglycerate kinase (PGK) in which the 'basic patch' residue His 62 has been replaced by a glutamine residue. Using 1H-NMR spectroscopy, it was found that 3-phosphoglycerate (3-PG) binding to the mutant protein induces the same conformational effects as for wild-type PGK, although the affinity was reduced by 2- to 3-fold. Kinetic studies show both Km for 3-PG and Vmax to be increased by approximately 2-fold relative to the wild-type enzyme. These data are consistent with the suggestion that His 62 assists in the binding of the substrate to the enzyme.

Histidine↗
PubMed1989

Site-directed mutagenesis of yeast phosphoglycerate kinase. The 'basic-patch' residue arginine 168.

There is evidence, some of it of questionable authenticity, which suggests that phosphoglycerate kinase takes up a more compact form following the binding of substrates. Using this evidence it has been assumed that a conformational rearrangement is required for phosphoryl transfer to occur and that this is brought about by moving the enzyme's two domains towards each other. In order to test this hypothesis we have modified, by site-directed mutagenesis, an arginine residue thought to be involved in stabilising the transition-state intermediate. Although some 1.3 nm away from the site of phosphoryl transfer, as seen in the crystallographically determined structure, the substitution of arginine 168 by lysine (R168K) more than halves the specific activity of the enzyme. Substituting the arginine with a methionine (R168M) reduces activity further, but not completely, thus proving that the charge associated with this residue is not essential for catalytic activity. Both mutations raise the Michaelis constants (Km) for ATP and glycerate 3-phosphate. The largest change is observed with the triose substrate and the methionine mutant, suggesting that the primary function of arginine 168 is to influence the environment of this substrate. The effect on activity of adding sulphate to R168K and R168M mutant enzyme has also been investigated. The sulphate activation effect at low substrate concentrations is reduced for the methionine substitution but almost abolished for the lysine substitution. The most reasonable explanation of all these findings is that, in the wild-type enzyme, the guanidinium group of arginine 168 forms a hydrogen bond with one of the triose substrate's C1 oxygens. This steric arrangement would not be possible in the 'open form' of this enzyme as observed in the crystal structure.

Arginine↗
PubMed1989

NMR analysis of site-specific mutants of yeast phosphoglycerate kinase. An investigation of the triose-binding site.

Site-specific mutants of yeast phosphoglycerate kinase have been produced in order to investigate the roles of the 'basic-patch' residues, arginine 168 and histidine 170. The fully-conserved residue, arginine 168, has been replaced with a lysine (R168K) and a methionine (R168M) residue, while the non-conserved histidine 170 has been replaced with an aspartate (H170D). Comparison of the 500-MHz 1H-NMR spectra of the mutant proteins with that of wild-type phosphoglycerate kinase shows that the overall fold of the mutants remains essentially unaltered from that of the native enzyme. Results of NOE experiments indicate that there are only very minor changes in structure in the vicinity of the mutations. These mutations have also led to firm sequence-specific resonance assignments to histidines 62, 167 and 170. NMR studies of 3-phosphoglycerate binding show that decreasing the positive charge in the sequence 168-170 reduces the binding of this substrate (by about 15-fold and 4-fold for mutants R168M and H170D respectively). Mutant R168K binds 3-phosphoglycerate with an affinity about twofold less than that of the native enzyme. Significantly, the activity of mutant H170D, measured at saturating substrate concentrations, is unchanged from that of the wild-type enzyme. This indicates that this residue is not of major importance in the binding or reaction of 3-phosphoglycerate. The observation is in agreement with results obtained for the wild-type enzyme, which indicate that 3-phosphoglycerate interacts most strongly with histidine 62 and least strongly with histidine 170, as would be predicted from the X-ray crystal structure. Substitution of positively charged arginine 168 with neutral methionine (or positively charged lysine) does not cause a detectable change in the pKa values of the neighbouring histidine groups, in as much as they remain below 3. The results reported here indicate that the observed reduction in catalytic efficiency relates less to direct electrostatic effects than to the mutants' inability to undergo 3-phosphoglycerate-induced conformational changes.

Arginine↗