The enzymes of the 3-hydroxyaspartate pathway have been found only in Micrococcus denitrificans growing on glyoxylate or a glyoxylate precursor. The partial purification of two of these enzymes (aspartate-glyoxylate transaminase and erythxo-beta-hydroxy- aspartate dehydratase) and the development of a specific assay for the third (erythro-beta-biydroxyaspartate aldolase) has allowed a study of their properties. All three were pyridoxal phosphate enzymes, although the aldolase alone exhibited a requirement for this coenzyme in crude extract, both for activity and stability. All three were most active at pH 7 or above, but only the aldolase and dehydratase had metal ion requirements, the former needing higher concentrations of divalent metal cations for activity than the latter. The purified aspartate-glyoxylate transaminase was specific for glyoxylate as amino acceptor, but reacted with a variety of amino donors, being most active with L-aspartate and L-serine. It was shown that these activities were associated with a single enzyme protein, and that this transaminase was responsible for the formation of glycine from glyoxylate in this system since glycine dehydrogenase activity was absent from extracts of glycollate-grown organisms. erythro-beta-Hydroxyaspartate aldolase was assayed by following pyruvate production from erythro-beta-hydroxy-beta-methyl-aspartate; the dehydratase, however, only acted on erythro-L-beta-hydroxyaspartate. The variation with pH of the Vmax and Km of the dehydratase suggested both that the substrate is bound in its most anionic form, and that the breakdown of the enzyme-substrate complex into products is governed by a dissociable group of pK'a about 8.5. The three enzymes of the pathway appeared to be co-ordinately induced and repressed. Succinate and malate, but not pyruvate or glucose, acted (perhaps indirectly) as repressors, and in addition glyoxylate appeared to have a direct inductive effect. Preliminary experiments suggested the absence of further control by inhibition or activation of individual enzymes in the pathway.

Thermophilic microorganisms present two fundamental proolems. (I) How are they able to grow at temperatures greatly above those normally considered suitable for life. (2) Why do thermophiles not grow at more moderate temperatures. This thesis is an account of experiments directed towards resolution of these problems using a prototrophic strain of the thermophile Bacillus stearothermophilus. Although virtually all the thermophile enzymes isolated to date have proved to be thermostable compared with their mesophile counterparts the possibility has remained open that part of the mechanism of thermophily is a rapid resynthesis of denatured proteins. It is shown here that protein turnover does occur during growth out the rate is too low to be compatible with this ''rapid repair" hypothesis. Studies of the stabilities of individual enzymes in intact cells confirm the results of the turnover experiments. This organisms' ability to grow on minimal media facilitated an examination of the energetics of growth. The energy required for growth is shown to be comparable with that of related mesophiles with no evidence for a massive diversion of energy for resynthesis of denatured macromolecules. These growth yield studies also show that the efficiency of substrate utilization decreases as the temperature rises. This is largely the result of changes in the extent of oxidation of the substrate. The effects of temperature on the regulation of a key enzyme, isolated from the thermophile, pyruvate kinase, have been studied to test the hypothesis that the temperature limits for growth of a particular microorganism are the result of an accumulation of derangements of metabolic regulation at temperature extremes. In most respects except thermostability the thermophile enzyme resembles those from mesophiles. Temperature effects on kinetic and regulatory parameters are too small to be implicated in the lower limit for growth but are probably involved in the temperature effects on growth yield.

Pyruvate carboxylase from Arthrobacter globiformis has been purified approximately 300-fold. The highly purified enzyme has a specific activity of 27.0 nmoles of oxaloacetate formed per mg of protein and gave two bands on SDS-polyacrylamide gel electrophoresis. The pyruvate caxboxylase containing band constituted approximately 80% of the total protein. The enzyme-catalysed reaction requires pyruvate, MgATP and HCO-3 and is activated by Mg2+ and K+ ions. The enzyme is inhibited by preincubation with avidin and by oxalate. The enzyme shows only about of its maximum activity in the absence of acetyl-CoA, which activates the enzyme with an A0.5 value of 25 muM. Activation by this effector is sigmoidal, with a Hill coefficient of about 3.0, and it is antagonised by L-aspartate, which has a Hill coefficient of between 2.0 and 4.0 depending on the concentration of acetyl-CoA. CoA is able to replace acetyl-CoA, with an A0.5 value of 1.3muM and a Hill coefficient of 3.0. Kinetic analysis of the reaction has shown its mechanism to be of the non-classical Ping Pong Bi Bi Uni Uni type with kinetically significant abortive complexes, enzyme-HCO-3-MgADP and enzyme-HCO-3-Pi. Apparent Km values and inhibition constants were determined for the substrates and product inhibitors of the enzyme. The enzyme was inactivated on incubation at 0°C or at pH 8.0, 15°C, with a change in s20,W value in each case which indicated that a halving of molecular weight was occurring. The molecular weight of the inactive form of the enzyme found at pH 8.0 was shown to be 290 000, implying a value of 580 000 for the molecular weight of the native enzyme. The subunit molecular weight of the enzyme was shown by SDS-polyacrylamide gel electrophoresis to be 130 000, suggesting that the native enzyme is tetrameric. This suggestion is supported by the fact that the sedimentation similar to that found for other pyruvate carboxylases which have been shown to be tetrameric. It has been shown that pyruvate carboxylase can exist in biotin-deficient A. globiformis in both apoenzyme and holoenzyme forms. The apoenzyme is converted to holoenzyme on addition of biotin to biotin-deficient cells, in the absence of protein synthesis. The total amount of pyruvate carboxylase in cells of A. globiformis appears to be determined largely by the nature of the carbon source used for growth. During growth at biotin concentrations of between 40 pg/ml and 1 ng/ml, the amount of pyruvate carboxylase holoenzyme appears to be directly dependent upon the concentration of biotin in the medium in cells grown on lactate, glycine and glucose. The results have been discussed by comparison with other pyruvate carboxylases. Kinetic and regulatory properties are broadly similar to those of other pyruvate carboxylases but the structural characteristics are markedly different from those of any other pyruvate carboxylases so far studied.

I Citrate synthase from Escherichia coli has been purified to homogeneity. Gel electrophoresis indicated that the enzyme exists in two or three electrophoretically separable forms in equilibrium with each other. Ultracentrifugation studies showed a size difference between these species: monomer (mol.wt. = 57,000), tetramer (mol.wt. = 230,000) and possibly a higher molecular weight protein. The tetramer is the predominant species, accounting for over 75 of the total protein at pH 8.0, and active-enzyme centrifugation demonstrated that it possesses catalytic activity. Electrophoresis in the presence of sodium dodecyl sulphate suggested that the subunits are of similar size with a molecular weight of approximately 55,000. Examination of the enzyme by electron microscopy after shadowing with platinum confirmed the predominance of the tetramer and further suggested that the subunits may be arranged in a square.;II Modification of the enzyme by photo-oxidation and by treatment with specific chemical reagents has been carried out to gain information on the amino acid residues involved in enzymic activity and in the inhibition of activity by NADH and ?-oxoglutarate. Several photo-oxidizable amino acids appear to be involved in activity. The nature of the pH-dependences of their rates of photo- -oxidation with Methylene Blue suggest that these are histidines, a conclusion supported by the greater rate of photo-inactivation with Rose Bengal. In addition, photo-oxidation suggests the participation of histidine at the ?-oxoglutarate effector site and cysteine at the NADH site. Amino acid analyses of native and photo-oxidized enzymes were consistent with these conclusions. Treatment of citrate synthase with diethyl pyrocarbonate and subsequent analysis of the modified enzyme confirmed the involvement of histidine in catalytic activity. Modification with 2-hydroxy-5-nitrobenzyl bromide also indicated the participation of tryptophan in the activity of the enzyme. Studies with the thiol-specific reagent DTNB were consistent with the photo-oxidation experiments in suggesting that cysteine residues are essential for NADH inhibition. Furthermore, the inactivation of the enzyme by DTNB indicated the additional involvement of cysteine in catalytic activity. A detailed kinetic analysis of the modification of the enzyme by DTNB is presented. The possible roles of these functional residues in the activity and regulation of citrate synthase are discussed.;III The effects of KCl on citrate synthase have been investigated. Examination of the enzyme by gel electrophoresis and gel filtration suggested that in the presence of KCl only the tetrameric species is present. Moreover, ultracentrifugation studies have demonstrated that the salt also induces the tetramer to assume a more compact conformation. Further evidence for these salt-induced changes has been gained from studies on the reactivity of cysteine residues to modification by DTNB and from an investigation of the exposure of tyrosine and tryptophan residues by solvent perturbation difference spectroscopy. Finally, preliminary kinetic studies indicated that the activation of citrate synthase by KCl is the result of an approximately 5-fold reduction in the Km for acetyl-CoA. The possible ways in which KCl and other neutral salts affect the structure of citrate synthase are discussed.;IV Preliminary investigations of the citrate synthases from two other Enterobacteriaceae (Serratia marcescens and Klebsiella aerogenes) have been made. The two enzymes have been purified and each shown to exist in an equilibrium between three electrophoretic species. Modification of these enzymes with DTNB has shown that, as with the citrate synthase of E.coli, thiols are required for full enzymic activity and sensitivity to NADH.

The activities of Acinetobacter lwoffi pyruvate dehydrogenase and succinate thiokinase have been shown to be under adenylate control. Pyruvate dehydrogenase was stimulated by AMP and ADP whereas ATP was inhibitory. The products NADH and acetyl-CoA inhibited the enzyme and AMP decreased the latter inhibition. On the other hand, A. Iwoffi succinate thiokinase utilised ADP (Km = l.l8mM), or GDP ( Km = 0.026mM), or IDP (Km = 0.023mM) as nucleotide substrate, and the GDP- or IDP- dependent activities were inhibited by ATP in the presence of nucleoside diphosphate kinase. Thus adenylate control of this enzyme may either be a consequence of the sensitive operation of the enzyme over the range in which the intracellular ADP concentrations may vary, or it may involve a nucleoside diphosphate kinase mediated interaction of ATP with the GDP or IDP pools. No adenylate control was observed with A. lwoffi aconitase, succinate dehydrogenase, fumarase and malate dehydrogenase. Therefore, these results together with those previously observed with citrate synthase, isocitrate dehydrogenase and ?-oxoglutarate dehydrogenase suggest that adenylate control of the citric acid cycle enzymes in A. lwoffi is exerted only on those enzymes which occur at the metabolic branch-points. This novel form of regulation in which several enzymes of a metabolic sequence are controlled by the same effector has been called "multipoint" control and presumably ensures that cycle activity 'is not limited by the withdrawal of intermediates. An examination of a large number of diverse bacterial species showed that the elements of the control are generally found together and were detected in all species of Acinetobacter examined, as well as in a few other Gram-negative strictly aerobic bacteria.

Four human IFNo: chromosomal genes have been isolated from a newly constructed placental DNA library in XL47. Restriction and sequencing analysis revealed that each gene had been described previously. However, one gene, SMTlll.lA, which encodes a full length IFN, is an allelic varient of a previously characterised pseudogene, thus indicating some degeneracy of the IFNa gene family. A chimaeric gene comprising the MuIFNx1 promoter (-188 to +52) and cat gene coding sequences has been constructed in vitro, enabling promoter function to be examined in mouse cells. Reproducible polyrI.rC mediated induction of CAT expression from the MuIFNx1 promoter has been demonstrated in pools of stably transfected, but not transiently transfected, L929 cells. Monitoring mRNA production revealed the transient accumulation of correctly initiated hybrid gene transcripts which precede optimum CAT production. Aspects of the structure/function relationship of the MuIFNx1 promoter have been investigated by oligonucleofide site directed mutagenesis. Comparative studies of IFNx promoter sequences identified prospective regulatory regions for mutagenesis. Quantitative CAT assays have been employed for promoter assessment. Additionally, the construction of a pseudogene comprising the wildtype MuIFNa1 promoter linked to an internally deleted cat gene has enabled both mutant and wildtype promoters functioning simultaneously in the same cell population to be assessed by S-1 nuclease protection studies using a common probe. Such studies have revealed three distinct cis-acting regions implicated in MuIFNx1 promoter function. Two are located upstream of the TATA box, defined by mutations at -87 and between -66 and -33 respectively. These reduce promoter activity 2 to 5 fold. The third, is defined by a mutation at +14, within the untranslated leader sequence. This enhances activity 2 to 3 fold. A deletion derivative of the MuIFNx1 promoter containing only 94bp of upstream sequence is inactive. Cis-activation by the Mo-MuSV enhancer restores inducibility to this promoter whereas the intact MuIFNx1 promoter is refractory to this element. This suggests that distinct cis-acting sequences dictate the efficiency and regulation of MuIFNx1 gene transcription.

Cloned genes, from Escherichia coli C, for the catabolism of homoprotocatechuate were available. The organisation and expression of these genes was investigated by a number of molecular genetic techniques. The exact position of the 5' end of one of the genes, hpcG, was determined by a combination of Southern blotting and DNA sequencing and subsequently used as a marker to aid the positioning and illucidation of the direction of transcription of the other genes. The genes were found to be arranged in two blocks of structural genes which were both transcribed in the same direction and a separate regulatory gene. Northern hybridisation analysis of RNA from wild type cells and from cells containing cloned genes indicated the presence of three transcripts of 4.5, 2.7 and 1.6 kbp. A model for the position of the hpc genes, their regulation and number of transcriptional units is proposed. One of the catalytic proteins, CHMS dehydrogenase, was purified and some of its properties investigated; homology between the amino-terminal amino acid sequence of this enzyme and the equivalent protein from Klebsiella pneumoniae M5a1 was very high. An oligonucleotide derived from shared protein sequence of the CHMS dehydrogenases from E. coli C and K, pneumoniae M5a1 was used to investigate homology in related species by Southern hybridisation. E. coli strains B and W DNA contained a hybridising fragment when tested at high stringency, but Pseudomonas putida DNA did not hybridise under these conditions. The same oligonucleotide was used to screen a K. pneumoniae M5a1 genomic library by colony hybridisation and clones carrying hpc genes were succesfully isolated. Initial analysis of these clones suggest that little gross homology exsists between the hpc genes of E. coli C and K. pneumoniae M5a1.

Purification of the DNA gyrase B protein consistently led to two contaminating bands of 47kDa and 43kDa molecular masses. These were found to be the C- and N-terminal fragments of GyrB. The specific supercoiling activity of GyrB was found to be consistently lower than the specific supercoiling activity of GyrA. This was due to about 90% of GyrB being in an uncoupled form. The uncoupled GyrB was found to have a relatively high ATPase activity, therefore an in-depth kinetic study on DNA gyrase was not possible. Kinetic studies were carried out on the 43kDa protein, which is a cloned N-terminal fragment of GyrB. The 43kDa protein was found to hydrolyse ATP at a relatively low rate, with 10 muM 43kDa having and apparent kcat of 0.01 s-1, and 20 muM a kcat of 0.02 s-1. A greater than first order dependence of rate upon 43kDa concentration was observed in the concentration range of 2-40 muM. Hyperbolic type kinetics were observed at constant 43kDa concentration (5, 10, 20 and 40 ?M) for the rate with respect to ATP concentration. A model which was found to be consistent with molecular weight studies and the kinetic data has been proposed. The 43kDa monomer can bind ATP but is not competent to hydrolyse ATP. Hydrolysis can only occur in the context of a 43kDa2ATP2 dimer, which leads to the collapse of the dimer into monomers and release of products. The rate limiting step at the protein concentration range used is the dimerisation step. Novobiocin and coumermycin inhibit the ATPase reaction, with novobiocin binding at a stoichiometry of 1 novobiocin molecule to 1 43kDa monomer and coumermycin binding with a stoichiometry of 1 molecule to two molecules of 43kDa protein. The inhibition by coumarin drugs appears non-competitive. ADPNP binding to the 43kDa protein was found to be slow, with a second order rate constant of 0.86 M-1s-1 to 9.9 M-1s-1. ADPNP seems to bind with stoichiometries varying from 2 per 43kDa dimer to 1 per 43kDa dimer. ATP and ADP inhibit the amount of ADPNP bound, with ATP having no effect on the rate of ADPNP binding and ADP decreasing the rate of ADPNP binding. Novobiocin and coumermycin inhibit ADPNP binding to the 43kDa protein. ADPNP dissociates from the 43kDa protein at a very slow rate, with a half life of about 8 days. ATP and ADP have little effect on this rate. However high concentrations of novobiocin (10-100 mM) dramatically increase the rate of ADPNP dissociation from the 43kDa protein, indicating different coumarin and nucleotide binding sites.

Mutants of three conserved residues in Dihydrofolate reductase (DHFR) from Lactobacillus casei have been studied to assess their roles in ligand binding and catalysis. Aspartate26 has been widely postulated to be the source of the proton in the reaction catalysed by DHFR; this was probed by the Aspartate26/Asparagine (D26N) mutation. The rate of hydride ion transfer, which governs kcat in the D26N mutant, is reduced by a factor of 10 (pH 7.5) when compared to the wild-type enzyme. The results argue against the role of Aspartate26 as the primary proton donor, but may be more consistent with a mechanism whereby it promotes enolisation of the substrate during the reaction. The Tryptophan21/Histidine (W21H) mutant binds the coenzyme NADPH over 1000-fold more weakly than wild-type DHFR. The magnitude of the negative cooperative effect between NADPH and FH4 (a crucial feature of the kinetic scheme of DHFR), has been greatly reduced in the W21H mutant, suggesting an important role for Tryptophan21 in the mechanism of negative cooperativity. The pH dependence of kcat for W21H (which is equivalent to the rate of hydride ion transfer) has a form that reflects the cooperative ionisation of two groups. Kinetic and NMR results suggest that the new Histidine21 is one of the groups responsible for the unusual ionisation curve. Substrate, inhibitor and coenzyme binding are unaffected by the Arginine57/Lysine (R57K) mutation. With the wild-type enzyme, loss of the ion-pair interaction between MTX and Arginine57 also leads to a loss of the interaction with Histidine28; this is not the case with the R57K mutant. The Arginine57/Lysine substitution has little effect on the rate of catalysis although the apparent pKa of kcat is reduced by 0.6 units, despite the site of catalysis being more than 15A away from the site of the mutation. The origin of this effect may be due to electrostatic or structural factors.

A Charomid ordered array library containing the 2-16kb size fraction of MboI-digested canine genomic DNA was screened with the multilocus probes, 33.6 and 33.15. Testing for polymorphism of the minisatellite loci in 48 resulting positive clones yielded seven polymorphic minisatellites with heterozygosities in the range 20-88%. Mendelian inheritance was confirmed shown for two of the polymorphic minisatellites. DNA fingerprinting studies of the level of inter- and intra-breed variation did not show any significant difference between the two. Analysis of intra-breed variation in Bedlington Terriers using two polymorphic minisatellites as single-locus probes revealed a significant reduction in the number of alleles in comparison with an agglomerated population sample, consistent with the high level of inbreeding within this breed. Multi-locus canine minisatellite probe analysis of unrelated species showed that related repeat sequences are present in numerous species. Use of single-locus canine minisatellite probes to analyse related canids suggested that polymorphic canine minisatellites are likely to show transience in their variability and detection, whereas monomorphic minisatellites are stable and more readily detected in related canids. Use of cCfaMP5, the most polymorphic canine minisatellite isolated to date, as a single-locus probe in paternity analysis demonstrates its applicability to forensic problems. Flanking sequence and partial repeat sequence data obtained for the minisatellite in cCfaMP5. The variable region in this minisatellite region is similar to many human minisatellites which show a distinct purine or pyrimidine strand bias. A mechanism whereby this minisatellite might have evolved is suggestedon the basis of the distribution and kinds of repeat units. An initial MVR-PCR analysis of CfaMP5 has been carried out and, with future optimization, it should be possible to digitally type canine minisatellite alleles, thereby widening the scope of the analysis of canine minisatellite variation.