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Biomedical subjects

T B Fitzpatrick

Publications and source records attributed to T B Fitzpatrick.

At least 19 recordsLinked to original sources
PubMed2001

Chorismate synthase from the hyperthermophile Thermotoga maritima combines thermostability and increased rigidity with catalytic and spectral properties similar to mesophilic counterparts.

Chorismate synthase, the last enzyme in the shikimate pathway, catalyzes the transformation of 5-enolpyruvylshikimate 3-phosphate to chorismate, a biochemically unique reaction in that it requires reduced FMN as a cofactor. Here we report on the cloning, expression, and characterization of the protein for the first time from an extremophilic organism Thermotoga maritima which is also one of the oldest and most slowly evolving eubacteria. The protein is monofunctional in that it does not have an intrinsic ability to reduce the FMN cofactor and thereby reflecting the nature of the ancestral enzyme. Circular dichroism studies indicate that the melting temperature of the T. maritima protein is above 92 degrees C compared with 54 degrees C for the homologous Escherichia coli protein while analytical ultracentrifugation showed that both proteins have the same quaternary structure. Interestingly, UV-visible spectral studies revealed that the dissociation constants for both oxidized FMN and 5-enolpyruvylshikimate 3-phosphate decrease 46- and 10-fold, respectively, upon heat treatment of the T. maritima protein. The heat treatment also results in the trapping of the flavin cofactor in an apolar environment, a feature which is enhanced by the presence of the substrate 5-enolpyruvylshikimate 3-phosphate. Nevertheless, stopped-flow spectrophotometric evidence suggests that the mechanism of the T. maritima protein is similar to that of the E. coli protein. In essence, the study shows that T. maritima chorismate synthase exhibits considerably higher rigidity and thermostability while it has conserved features relevant to its catalytic function.

Bacterial Proteins↗
PubMed2001

Structure and characterization of the glycan moiety of L-amino-acid oxidase from the Malayan pit viper Calloselasma rhodostoma.

Ophidian L-amino-acid oxidase (L-amino-acid oxygen:oxidoreductase, deaminating, EC 1.4.3.2) is found in the venom of many poisonous snakes (crotalids, elapids and viperids). This FAD-dependent glycoprotein has been studied from several snake species (e.g. Crotalus adamanteus, Crotalus atrox and Calloselasma rhodostoma) in detail with regard to the biochemical and enzymatic properties. The nature of glycosylation, however, as well as the chemical structure(s) of the attached oligosaccharide(s) are unknown. In view of the putative involvement of the glycan moiety in the biological effects of ophidian L-amino-acid oxidase, notably the apoptotic activity of the enzyme, structural knowledge is needed to evaluate its exact function. In this study we report on the glycosylation of L-amino-acid oxidase from the venom of the Malayan pit viper (Calloselasma rhodostoma). Its glycosylation is remarkably homogeneous with the major oligosaccharide accounting for approximately 90% of the total sugar content. Based on detailed analysis of the isolated oligosaccharide by 2D NMR spectroscopies and MALDI-TOF mass spectrometry the glycan is identified as a bis-sialylated, biantennary, core-fucosylated dodecasaccharide. The biological significance of this finding is discussed in light of the biological activities of the enzyme.

Amino Acid Oxidoreductases↗
PubMed1999

13C NMR study of how the oxyanion pKa values of subtilisin and chymotrypsin tetrahedral adducts are affected by different amino acid residues binding in enzyme subsites S1-S4.

A range of substrate-derived chloromethane inhibitors have been synthesized which have one to four amino acid residues. These have been used to inhibit both subtilisin and chymotrypsin. Using 13C NMR, we have shown that all except one of these inhibitors forms a tetrahedral adduct with chymotrypsin, subtilisin, and trypsin. From the pH-dependent changes in the chemical shift of the hemiketal carbon of the tetrahedral adduct, we are able to determine the oxyanion pKa in the different inhibitor derivatives. Our results suggest that in both the subtilisin and chymotrypsin chloromethane derivatives the oxyanion pKa is largely determined by the type of amino acid residue occupying the S1, subsite while binding in the S2-S4 subsites only has minor effects on oxyanion pKa values. Using free energy relationships, we determine that the different R groups of the amino acid residues binding in the S1 subsite only have minor effects on the oxyanion pKa values. We propose that the lower polarity of the chymotrypsin active site relative to that of the subtilisin active site explains why the oxyanion pKa is higher and more sensitive to the type of chloromethane inhibitor used in the chymotrypsin derivatives than in the subtilisin derivatives.

Alkylation↗
PubMed1998

The effect of histidine-228 on the catalytic efficiency and stereospecificity of the serine hydroxymethyltransferase catalysed exchange of the alpha-protons of amino acids.

13C-NMR has been used to determine how replacing the histidine-228 residue of serine hydroxymethyltransferase (EC 2.1.2.1) by an asparagine residue effects the catalysis of the hydrogen-deuterium exchange of the alpha-protons of [2-13C]glycine at pH 7.8. The H228N mutation did not lead to a large change in the stereospecificity of the first order exchange rates of the alpha-protons of glycine both in the presence and in the absence of tetrahydrofolate. However, the mutation did lead to large decreases in the stereospecificity of the second order exchange rate in both the presence and the absence of tetrahydrofolate. In the absence of tetrahydrofolate this decrease in stereospecificity was largely due to the decrease in the second order exchange rate of the pro-2S proton, while in the presence of tetrahydrofolate the large increase in the second order exchange rate of the pro-2R proton of glycine made a major contribution. We conclude that the H228N mutation has significant effects on the catalytic efficiency and stereospecificity of the second order exchange reactions, but only a small effect on the corresponding first order exchange reactions.

Asparagine↗
PubMed1998

The pyridoxal-5'-phosphate-dependent catalytic antibody 15A9: its efficiency and stereospecificity in catalysing the exchange of the alpha-protons of glycine.

13C-NMR has been used to follow the exchange of the alpha-protons of [2-(13)C]glycine in the presence of pyridoxal-5'-phosphate and the catalytic antibody 15A9. In the presence of antibody 15A9 the 1st order exchange rates for the rapidly exchanged proton of [2-(13)C]glycine were only 25 and 150 times slower than those observed with tryptophan synthase (EC 4.2.1.20) and serine hydroxymethyltransferase (EC 2.1.2.1). The catalytic antibody increases the 1st order exchange rates of the alpha-protons of [2-(13)C]glycine by at least three orders of magnitude. We propose that this increase is largely due to an entropic mechanism which results from binding the glycine-pyridoxal-5'-phosphate Schiff base. The 1st and 2nd order exchange rates of the pro-2S proton have been determined but we were only able to determine the 2nd order exchange rate for the pro-2R proton of glycine. In the presence of 50 mM glycine the antibody preferentially catalyses the exchange of the pro-2S proton of glycine. The stereospecificity of the 2nd order exchange reaction was quantified and we discuss mechanisms which could account for the observed stereospecificity.

Antibodies, Catalytic↗
PubMed1998

A substrate-induced change in the stereospecificity of the serine-hydroxymethyltransferase-catalysed exchange of the alpha-protons of amino acids--evidence for a second catalytic site.

NMR has been used to study the catalysis of the hydrogen-deuterium exchange of the alpha-protons of amino acids by serine hydroxymethyltransferase (EC 2.1.2.1) from Escherichia coli. 13C-NMR was used to follow the exchange of the alpha-protons of [2-13C]glycine. The enzyme-catalysed first-order exchange rate of the pro-2S proton of glycine was approximately 7000 times more efficient than that of the pro-2R proton of glycine at both pH 7.0 and 7.8. 1H-NMR was used to follow the hydrogen-deuterium exchange rates of the alpha-protons of L- and D-2-amino derivatives of butyric, pentanoic and hexanoic acids at pH 7.8. Increasing the size of the R-group leads to a progressive change in the stereospecificity of the exchange reaction from the pro-2S proton of glycine to the 2R proton of L-amino acids. The stereospecificity for the alpha-protons of L-amino acids increased as the size of the R-group increased. With glycine, removal of tetrahydrofolate led to a large decrease in the stereospecificity of the exchange reaction but did not affect the exchange rates of the alpha-protons of any of the larger amino acids studied. We show that the Schiff base formed between L-2-aminohexanoic acid (L-norleucine) and pyridoxal 5'-phosphate binds at a different site from the Schiff base between glycine and pyridoxal 5'-phosphate. The molecular basis of these results is discussed.

Amino Acids↗
PubMed1998

The benefits and risks of long-term PUVA photochemotherapy.

In 1974 a new photobiologic principle i.e. light + drug, called photochemotherapy was discovered in Boston and immediately confirmed in Vienna. Psoralen + UVA (PUVA) photochemotherapy has now been applied to the treatment of more than 24 heterogeneous groups of diseases, especially psoriasis and mycosis fungoides. After 24 years of experience in thousands of patients with psoriasis and 23 other skin disorders, virtually the only risk is the development of squamous-cell carcinomas. This risk is low with two exceptions: previous history of treatment with ionizing radiation or inorganic trivalent arsenic, and patients with recalcitrant psoriasis who require continuous treatment for many years. In a recent report from a large USA clinical trial, melanoma developed in a few patients with psoriasis treated with PUVA. This prospective clinical trial did not have a control population, and therefore, the conclusion that PUVA can cause melanoma is tentative.

Clinical Trials as Topic↗
PubMed1995

Modifications of PUVA.

The goal of photochemotherapy in psoriasis is to attempt to lower the number of exposures and the total cumulative doses while still maintaining good control of the disease. PUVA has been modified by using better psoralen preparations and more effective light sources, and it also has been combined with other treatment modalities. The objectives in modifying PUVA and combining PUVA with other treatment modalities are to increase efficacy, to reduce short- and long-term adverse effects, and to reduce the cost of treatment. Modalities that have been combined with PUVA include topical corticosteroids, anthralin, calcipotriene ointment, methotrexate, UVB, retinoids, and cyclosporine.

Costs and Cost Analysis↗