Nitric oxide synthase (NOS) catalyzes nitric oxide (NO) production in a two step reaction. The first step involves L-arginine being oxidised to N^G-hydroxy-L-arginine (NOHA) that remains bound to the enzyme before it is oxidised to NO and L-citrulline in the second step. The three mammalian isozymes are homodimeric enzymes and each subunit is composed of a reductase and an oxygenase domain. The oxygenase domain contains the arginine binding site, one cysteine ligated heme thiolate, and one H₄B molecule ((6R)-5,6,7,8-Tetrahydrobiopterin). The enzyme’s substrates are O₂, L-arginine (or NOHA) and NADPH. H₄B is an essential cofactor for NO production by NOS and its roles are in dimerization and as redox cofactor. The mechanism of the reaction between L-arg and oxygen in the active site is currently uncertain. Therefore in order to alter the course of the reaction site-directed mutagenesis was conducted: Glycine 586 of nNOS was replaced by a serine residue (G586S nNOS_oxy) and expressed and purified from <i>E.coli</i>. Stopped flow kinetic experiments showed the formation of a novel reaction intermediate during G586S nNOS_oxycatalysis in the presence of H₄B and substrate, subsequent to the formation of the oxy-ferrous compound. It is suggested that the new intermediate is formed as a transient along the path of the NO production reaction and has spectroscopic resemblance to the putative P450 active species, the oxy-ferryl compound. Crystals of the G586S mutant have been obtained and the solved x-ray structure shows the newly introduced serine residue pointing toward the guanidinium group of L-arg, reinforcing its involvement in the stabilization of a reaction intermediate.

Flavocytochrome <i>b</i>₂ from the yeast <i>Saccharomyces cerevisiae </i>is a L-lactate:cytochrome <i>c</i> oxidoreductase. The crystal structure of this homotetrameric enzyme has been solved to 2.4 Å. Each subunit consists of two distinct domains; a small (100 residue) N-terminal cytochrome domain containing a <i>b</i>-type heme, and a larger (411 residue) C-terminal domain containing flavin mononucleotide (FMN). The two domains are connected by a hinge sequence, running from residues 89 to 103. It has been proposed that the most likely role of this hinge region is to confer inter-domain mobility, allowing movement of the cytochrome domain with respect to the flavin domain. In this work a disulphide-bridge was engineered in a position, such that the two domains would be fixed in close proximity. Site-directed mutagenesis was used to make the double mutation, N42C:K324C. The crystal structure of the N42C:K324C mutant enzyme was solved to 2.8 Å resolution. An inspection of this structure has confirmed the existence of the imposed disulfide-bridge. In addition, the four <i>b</i>₂-heme domains of the tetramer are ordered, indicating their limiting mobility. Steady-state kinetic analyses with L-lactate, using ferricyanide [Fe(CN)₆]^3- and cytochrome <i>c</i> as electron acceptors were carried out. The formation of the disulfide-bridge causes a 15-fold decrease in <i>k</i>_cat with both electron acceptors. Since [Fe(CN)₆]^3- can accept electrons from both the FMN and <i>b</i>₂-heme while cytochrome <i>c</i> can only accept electrons from the <i>b</i>₂-heme this indicates that it is the rate of FMN reduction by L-lactate that is primarily affected by disulphide-bridge formation. Pre-steady-state kinetic analyses with L-lactate are consistent with the steady-state data. The formation of the disulphide-bridge makes it impossible to measure the rate constant for FMN reduction directly while <i>b</i>₂-heme reduction shows a rate constant some 450-fold less than in the open. If flavin to <i>b₂</i>-heme electron transfer is much faster then <i>b</i>₂-heme reduction will be limited by the rate of formation of reduced flavin. Thus, disulfide- bridge formation substantially lowers the rate of FMN reduction.

Nuclear pre-mRNA splicing occurs within a multicomponent complex, the spliceosome, which is formed by the ordered assembly of small nuclear ribonucleoprotein particles (snRNPs), and a host of other protein factors. The PRP2 protein of <i>Saccharomyces cerevisiae</i> is a non-snRNP factor required for the first cleavage-ligation step of the nuclear pre-mRNA splicing reaction. It is not required for early stages of complex assembly, but associates transiently with spliceosomes prior to and throughout step 1. PRP2 is a member of the DEAD/DEAH box family of putative RNA helicases and has been shown to possess RNA-dependent ATPase activity. The highly transient association of PRP2 with spliceosomes has hindered biochemical studies of the interactions of this protein, and therefore a genetic approach, which has been used successfully to study protein-protein interactions in other systems, was adopted. The principle behind this approach is that mutations in the <i>PRP2</i> gene that confer a dominant negative phenotype (ie. causing a dominant inhibitory effect over the wild-type) may do so by preventing release of PRP2 from splicing complexes. Stalled spliceosomes containing non-functional PRP2 would accumulate, facilitating genetic and biochemical analyses of this interaction. From a pool of randomly generated <i>PRP2</i> mutants, one dominant negative allele, <i>PRP2-dn1</i>, was isolated that caused a decline in cell growth rate when overexpressed in a wild-type yeast strain. Cell growth inhibition was presumably the result of the observed defect in pre-mRNA splicing, and was partially alleviated by simultaneous co-overexpression of wild-type <i>PRP2</i>.

Flavocytochrome <i>c</i>₃ (Fc<i>c</i>₃) is a 63.8 kDa soluble fumarate reductase produced by the bacterium <i>Shewanella frigidimarina.  </i>The aim of this project was to investigate the ability of Fc<i>c</i>₃ to reduce alternative substrates, particularly those that may result in the production of chiral molecules. Fc<i>c</i>₃ is able to reduce 2-methylfumarate to 2-methylsuccinate <i>in vitro</i>, with a k_cat of 8.97.s^-1 ± 0.43, and a K_m of 31.7 μM ± 8.5 at pH 7.2. Circular dichroism spectroscopy revealed that reduction of 2-methylfumarate by wild-type Fc<i>c</i>₃is stereospecific, producing S-methylsuccinate at >95% enantiomeric excess. The crystal structure of Wild-type Fc<i>c</i>₃ with 2-methylfumarate bound was solved to 1.5 Å and showed that the mode of 2-methylfumarate binding to the active site always results in the production of S-methylsuccinate. A range of fumarate derivatives were tested as potential substrates but wild-type Fc<i>c</i>₃ did not catalyse the reduction of anything other than fumarate and 2-methylfumarate. In order to find out if the substrate specificity of Fc<i>c</i>₃ could be altered, active site residues involved in Hydrogen-bonding with substrate were substituted by site-directed mutagenesis. Subsequent kinetic studies demonstrated that most of the mutants constructed were still able to reduce fumarate and 2-methylfumarate, although at rates differing from that measured for wild-type Fc<i>c</i>₃. Crystal structures were obtained for the mutants T377A and H365G at 2.0 Å and 1.9 Å respectively. Previous theoretical modelling studies had predicted that some mutants (especially H365G) may be able to catalyse the reduction of some mono-acids. However, it appears that this is not the case as none of the engineered forms of Fc<i>c</i>₃ were able to reduce any substrates other than fumarate and 2-methylfumarate. Fc<i>c</i>₃ is therefore a highly substrate-specific fumarate reductase.

No description available.

The biohydroxylation potential of the mutant enzyme, cytochrome P-450 cam Y96A monooxygenase (Y96A), has been investigated with a series of substrates which differ structurally from that of the natural substrate, D-(+)-camphor. Design of substrates essentially consisted of coupling an aromatic side-chain with an alicyclic moiety <I>via</I> an ester, ether or amide link. Assays have been performed with Y96A in order to obtain data on key factors of the biohydroxylation reaction such as substrate binding and turnover to give hydroxylated products. Y96A research has been complemented by a thorough investigation of the biohydroxylation capability of bacterium, <I>Rhodococcus rhodochrous</I> NCIMB 9703. Compounds found to be inactive with Y96A were reacted with this whole cell system, and positive results have been achieved in all cases.

The biotin biosynthetic enzyme 8-amino-7-oxononanoate synthase (AONS) from <i>Escherichia coli </i>is an homodimeric enzyme and relies on a pyridoxal-5'-phosphate (PLP) cofactor for catalytic activity. Specific activity of the over-expressed enzyme is approximately 0.4U at 30°C. To investigate the role of residues at the active site on the catalytic mechanism of the enzyme, two separate mutants have been created by site directed mutagenesis. H133 lies in parallel with the pyridine ring of the cofactor, and was mutated to a phenylalanine residue, with subsequent loss of cofactor binding. Alteration of the lysine 236 residue crucial for forming an aldimine linkage with the cofactor, to alanine resulted in a reduction, but no loss, of cofactor binding. The specific activities of both mutants were reduced by approximately ten-fold. The corresponding <i>bio</i>F gene from an hyperthermophilic bacterium, <i>Aquifex</i> <i>aeolicus</i>, was cloned and AONS over-expressed in an <i>E. coli</i> host. The enzyme was characterised by a number of biophysical techniques. It is a dimer in solution, with a monomeric mass of 42.3 kDa and one active site per subunit. Specific activity of the purified enzyme was 0.7U at 30°C, and optimum activity occurred at 80°C. The heat stable enzyme displays a greater affinity for its PLP cofactor and the binding of the coenzyme on mutation of the active site histidine residue corresponding to H133 in <i>E. coli</i>, indicates that PLP is bound by a residue not involved in <i>E. coli</i> AONS-PLP binding. Crystals grew in wells containing 1.6M-2.5M ammonium sulphate at 60°C produced orthorhombic crystals, which diffracted to 2.94Å. Analysis of the diffraction data suggested a space group of P2₁2₁2and a large unit cell containing at least 6 subunits in the asymmetric unit. Phasing was achieved by molecular replacement using the structure of the <i>Bacillus sphaericus </i>AONS protein.

In this thesis, the two ORFs believed to code for <I>pogo</I> transposase were joined in frame by PCR and cloned into a pGEX vector. The <I>pogo</I> transposase was fused to the C-termini of glutathione-S-transferase (GST) and expressed in <I>E. coli</I>. The DNA binding activity of <I>pogo</I> transposase was tested by gel retardation assays in the presence of specific and non-specific competitors. <I>pogo</I> transposase was shown to be able to bind specifically to the end sequences of the element. The transposase binding sites within the <I>pogo</I> element were identified by testing the transposase binding ability of deleted end sequences of the element. A 12 base pair consensus sequence was found and shown to be responsible for binding to the transposase. There are several copies of the 12 bp transposase binding site located near each end, in the 5' subterminal region, and in the middle of the element respectively. The binding sites at the ends might be involved in forming the synaptic DNA-protein complex and others in regulating transposition. The DNA binding domain of <I>pogo</I> transposase was identified by expressing different regions of the transposase and determining their sequence specific DNA binding ability. The DNA binding domain has been shown to be located in the N-terminal 75 amino acid region. Site direct mutagenesis was used to study the role of the predicted helix-turn-helix (HTH) motif located within the DNA binding domain. Substitution of the positively charged basic amino acids within the recognition helix by alanine abolished the DNA binding activity of the protein. Substitutions introducing prolines into the first or second helices to disrupt their structures also greatly reduced the DNA binding activity of the protein. These data support the idea that the HTH motif in the DNA binding domain is responsible for the specific DNA binding activity of the transposase.

Flavocytochrome <i>b</i><SUB>2</SUB> (<i>b</i><SUB>2</SUB>) from baker's yeast catalyses the two electron oxidation of L(&43 )lactate to pyruvate and subsequent reduction of cytochrome c. The enzyme is a tetramer of identical subunits, each of which consists of two functionally distinct domains; a flavodehydrogenase domain containing flavin mononucleotide (FMN) and a cytochrome domain containing protoheme IX. The work in this thesis describes the investigation of the roles of specific amino acid residues, carried out through the kinetic and spectroscopic characterisation of site-directed mutant forms of <i>b</i><SUB>2</SUB>. Tyr143 is an active site residue which lies between the flavodehydrogenase and cytochrome domains of <i>b</i><SUB>2</SUB>. Its role in the catalytic cycle was examined by replacement of this residue with phenylalanine. The most significant effect of this mutation was a change in the rate-limiting step of catalysis. In the wild-type enzyme, this is abstraction of the lactate C2-H. In the mutant enzyme, interdomain electron transfer between the flavin and heme prosthetic groups is now the slowest step. The rate of heme reduction by lactate, determined using the stopped-flow method, is decreased by > 20-fold from 445 ± 50 s^-1 in the wild type enzyme to 21 ± 2 s<SUP>-1</SUP> in the mutant enzyme. Decreases in kinetic isotope effects seen with [2-<SUP>2</SUP>H]lactate for the mutant enzyme compared to the wild-type, both for flavin and for heme reduction, also provide support for a change in the nature of the rate-limiting step. Other kinetic parameters are all consistent with the mutation having a dramatic effect on inter domain electron transfer. It therefore appears that Tyr143 plays a key role in facilitating electron transfer between the flavin and heme groups. Further studies on the role of Tyr143 were carried out by replacing this residue with glutamine. Kinetic results show that the enzyme is a poor lactate dehydrogenase. Furthermore, the lactate K<SUB>M</SUB> has increased by > 100-fold from 0.5mM in the wild-type enzyme to -65mM in the mutant enzyme. These and other kinetic results suggest that lactate to flavin electron transfer is the slowest step in catalysis, due to conformational effects at the active site. This is consistent with circular dichroism studies which show that a significant flavin/aromatic residue interaction has been lost. Two other interdomain residues, Phe325 and Tyr97, were investigated by substitution with alanine and tyrosine respectively. Although kinetic results indicate that Tyr97 is not of great functional significance, it is shown that Phe325 contributes towards domain/domain integrity and recognition.

No description available.