Biomedical subjects

M A Phillips

Publications and source records attributed to M A Phillips.

At least 37 records · Page 2Linked to original sources

PubMed1999

The effects of the novel anxiolytic drug lesopitron, a full and selective 5-HT1A receptor agonist, on pupil diameter and oral temperature in man: comparison with buspirone.

We investigated the effects of two 5-HT1A receptor agonists, buspirone and lesopitron, upon pupil size in human volunteers at an ambient luminance level of 32 Cd m(-2) and in darkness. Pupil diameter was monitored with a binocular infrared television pupillometer, before and after the administration of treatments for 4 h at 20-min intervals. Two experiments were conducted. In Experiment 1, 14 healthy male volunteers participated in seven weekly sessions, each associated with the ingestion of one capsule (buspirone 5, 10 and 20 mg, lesopitron 10, 20 and 40 mg and placebo), according to a double-blind balanced, cross-over design. Both buspirone and lesopitron tended to decrease pupil diameter. In darkness, only the highest dose of buspirone (20 mg) caused a miosis that was statistically significant. However, at the luminance level of 32 Cd m(-2) buspirone 10 and 20 mg evoked statistically significant miotic effects, as did the highest dose of lesopitron (40 mg). The miotic effect was significantly greater at 32 Cd m(-2) than in darkness after each dose of buspirone and the highest dose (40 mg) of lesopitron. In Experiment 2, pupil diameter and oral temperature were monitored with an electronic thermometer at 40-min intervals. Twenty healthy male volunteers participated in two weekly sessions, each associated with the sublingual application of 100 microl hydroalcoholic solution (lesopitron 20 mg, placebo), according to a double-blind balanced cross-over design. Lesopitron caused a significant miosis both in darkness and at the luminance level of 32 Cd m(-2); the miosis was greater at 32 Cd m(-2) than in darkness. Lesopitron tended to decrease oral temperature; this effect however, was not statistically significant. The greater effectiveness on the pupil of lesopitron administered sublingually in a solution indicates the importance of first-pass metabolism in reducing the effectiveness of the drug when administered by the mouth. The miosis observed in both experiments may be due to either a sympatholytic or a parasympathomimetic effect of the drugs, or both. The light-dependence of the miosis indicates that the 5-HT1A receptor agonists can modulate the light reflex, possibly via the noradrenergic control of central cholinergic neurones in the Edinger-Westphal nucleus.

Adult↗

PubMed1998

Carbon-13 isotope effect studies of Trypanosoma brucei ornithine decarboxylase.

Carbon isotope effect studies were undertaken with the wild-type pyridoxal 5'-phosphate (PLP)-dependent enzyme ornithine decarboxylase (ODC) from Trypanosoma brucei and with several active site mutants of the enzyme. For the decarboxylation of the optimal substrate, L-ornithine, by wild-type ODC, the observed carbon isotope effect (k12/k13) is 1.033 at pH 7.3. In comparison to the expected intrinsic isotope effect (k12/k13 = 1.06) for decarboxylation, this value suggests that both the rate of decarboxylation and the rate of Schiff base interchange with L-ornithine are partially rate-limiting for the reaction steps up to decarboxylation. In contrast, with the alternate substrate L-Lys, which shows lower catalytic efficiency, the carbon isotope effect increased to 1.063, demonstrating that decarboxylation has become the rate-limiting step. For the mutant enzymes, E274A ODC and C360A ODC, with L-ornithine as substrate the carbon isotope effect also approaches the intrinsic limit. Glu-274 was previously demonstrated to play a direct role in carbanion stabilization, and thus the large carbon isotope effect (k12/k13 = 1.055) is consistent with an impaired rate of decarboxylation compared to wild-type ODC. In contrast, for K69A ODC, the isotope effect is almost entirely suppressed, suggesting that Schiff-base formation (which now must occur from enzyme-bound PLP, rather than from an enzyme-bound PLP-Schiff base) has become rate-determining.

Animals↗

PubMed1998

Trypanosoma brucei gamma-glutamylcysteine synthetase. Characterization of the kinetic mechanism and the role of Cys-319 in cystamine inactivation.

The parasitic protozoan Trypanosoma brucei utilizes a conjugate of glutathione and spermidine, termed trypanothione, in place of glutathione to maintain cellular redox balance. The first committed step in the biosynthesis of glutathione and thereby trypanothione, is catalyzed by gamma-glutamylcysteine synthetase (gamma-GCS). We have determined the kinetic mechanism for T. brucei gamma-GCS. The kinetics are best described by a rapid equilibrium random ter-reactant mechanism, in which the model derived Kd values for the binding of L-Glu, L-alpha-aminobutyrate, and ATP to free enzyme are 2.6, 5.1, and 1.4 mM, respectively. However, significant dependences exist between the binding of some of the substrate pairs. The binding of either ATP or L-Glu to the enzyme increases the binding affinity of the other by 18-fold, whereas the binding of L-Glu or L-alpha-aminobutyrate decreases the binding affinity of the other by 6-fold. Similarly to the mammalian enzyme, cystamine is a time-dependent, irreversible inhibitor of T. brucei gamma-GCS. It has been suggested by several studies that cystamine labels an active site Cys residue essential for catalysis. Among the enzymes reported to be inactivated by cystamine, only one Cys residue is invariant (Cys-319 in T. brucei gamma-GCS). Mutation of Cys-319 to Ala in T. brucei gamma-GCS renders the enzyme insensitive to cystamine inactivation without significantly affecting the enzyme's catalytic efficiency, kinetic mechanism, or substrate affinities. These studies suggest that cystamine inactivates the enzyme by blocking substrate access to the active site and not by labeling an essential active site residue.

Amino Acid Sequence↗

PubMed1998

Application of an almost traceless linker in the synthesis of 2-alkylthiobenzimidazole combinatorial libraries.

Resin-bound triphenylphosphine was coupled to 4-fluoro-3-nitrobenzyl bromide, and 2-alkylthiobenzimidazoles were synthesized on resin in 4 steps using standard chemistries. Cleavage of the compounds from the resin was achieved with 10% NaOH in MeOH to leave a methyl group at the attachment point. A total of 47 amines and 40 electrophiles were evaluated, defining the scope of the reactions, culminating in the synthesis of an 80-member test library of high purity as determined by HPLC.

Alkanes↗

PubMed1998

Modulation of human epidermal cell response to 2,3,7,8-tetrachlorodibenzo-p-dioxin by epidermal growth factor.

Cultured human epidermal cells were treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the presence or absence of epidermal growth factor (EGF). In both normal keratinocytes and a spontaneously immortalized keratinocyte (SIK) line, TCDD treatment in the absence of EGF induced a marked reduction in colony size and cell number, and it perturbed colony morphology. These effects were largely prevented by EGF, indicating that growth factor action in the cellular microenvironment may considerably modify TCDD action in target cells. Both TCDD and EGF substantially reduced expression of the differentiation markers keratin 1 and keratin 10 in the normal and immortalized cells, and did so in an additive fashion. The cells did not display a general loss of differentiated function, since several other markers, including involucrin, were little affected. EGF dramatically stimulated telomerase activity in SIK cultures, and TCDD prevented this action but not by reducing cell growth. However, EGF did not stimulate telomerase activity in normal human epidermal cells despite an evident increase in their growth. The growth factor stimulation of telomerase in the minimally deviated SIK line suggests that derepression of enzyme activity in normal cells may occur in a stepwise fashion during neoplastic progression. TCDD could act as a late stage tumor promoter by selecting for variants in which telomerase is constitutively active.

Cell Differentiation↗

PubMed1997

Characterization of the reaction mechanism for Trypanosoma brucei ornithine decarboxylase by multiwavelength stopped-flow spectroscopy.

Ornithine decarboxylase (ODC), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, catalyzes the first committed step in the biosynthesis of polyamines. The UV-visible spectra of PLP (300-500 nm) was used to monitor the formation and breakdown of ODC reaction intermediates by multiwavelength stopped-flow spectroscopy to determine the reaction mechanism. Global kinetic analysis of the spectral data acquired after mixing ODC with saturating substrate (S) or product (P) (10 mM ornithine or 10 mM putrescine at 4 degrees C) suggests that ODC-catalyzed decarboxylation proceeds by the following reaction mechanism: ODC + S if A --> B --> C --> D --> E/F if ODC + P, where A-F are intermediates along the reaction path. Species B, which has absorbance maxima of 350 and 450 nm, is spectrally distinct from the other intermediates. On the basis of the calculated spectral characteristics, species B is likely to represent a quinoid intermediate which would be formed directly upon decarboxylation of ornithine. Thus, the data suggest that the reaction proceeds via formation of a Schiff base intermediate (species A) during the dead time of the stopped-flow instrument, followed by formation of a quinoid intermediate with a rate constant of 21 s-1. The quinoid intermediate decays in two steps (with rates of 145 and 1.0 s-1, respectively) to a Schiff base with putrescine (species D). Protonation of the Calpha carbon is required for the formation of species D, suggesting that the first of these events represents this step. The decay of species D to free enzyme and product occurs via a minimum of two intermediates and at an overall rate constant of 1-3 s-1. By comparison to the steady-state turnover number (kcat = 0.5 s-1 at 4 degrees C), these data identify product release as a rate-determining step in the overall reaction.

Animals↗

PubMed1997

Role of Arg-277 in the binding of pyridoxal 5'-phosphate to Trypanosoma brucei ornithine decarboxylase.

The pyridoxal 5'-phosphate (PLP) binding site in Trypanosoma brucei ornithine decarboxylase (ODC) has been studied by site-directed mutagenesis and spectroscopy. The beta/alpha barrel model proposed for the eukaryotic ODC structure predicts that the phosphate group of PLP is stabilized by interactions with a Gly-rich loop (residues 235-237) and by a salt bridge to Arg-277 [Grishin, N. V., Phillips, M. A., & Goldsmith, E. J. (1995) Protein Sci. 4, 1291-1304]. Mutation of Arg-277 to Ala increases the K(m) for PLP by 270-fold compared to that of wild-type ODC while reducing k(cat) by only 2-fold at pH 8. PLP binding affinity was measured directly by ultrafiltration; the K(d) for PLP is at least 20-fold higher in the mutant enzyme at pH 8. In addition, R277A ODC also has weaker binding affinities for a series of cofactor analogs than the wild-type enzyme. These results demonstrate that Arg-277 is necessary for high-affinity PLP binding by ODC. The 31P NMR spectra of ODC suggest that the phosphate is bound in a strained conformation as a dianion to both wild-type and R277A ODC. However, the 31P chemical shift for R277A ODC (6.7 ppm) is 0.5 ppm downfield from that observed for the wild-type enzyme, indicating that the environment of the enzyme-bound phosphate is altered in the mutant enzyme. The binding affinity of PLP for both wild-type and R277A ODC is weaker at high pH, corresponding to the titration of a protonated species with a pK(a) of approximately 8.5. Concomitant with these changes are a decreased k(cat) and an altered absorption spectra which arises from bound PLP. PLP bound to wild-type ODC has a 31P chemical shift and a CD signal observable over the entire tested pH range (7-9). In contrast, for R277A ODC between pH 8 and 9, the 31P chemical shift becomes solution-like and the CD signal is abolished. The data suggest that for R277A ODC the rigid PLP binding mode which characterizes the wild-type enzyme is lost at high pH. Thus, multiple interactions between the wild-type active site and PLP maintain the cofactor in a constrained conformation that is essential for efficient catalysis, tempering the consequence of the removal of any single interaction.

Animals↗

PubMed1997

Trypanosoma brucei: effects of methoprene and other isoprenoid compounds on procyclic and bloodstream forms in vitro and in mice.

Drug therapy for the treatment of African sleeping sickness is limited by toxicity and resistance and in the last 50 years only one new drug has been introduced for the treatment of the human disease. We report that the juvenile hormone analog, methoprene, and several structurally related isoprenoid compounds kill Trypanosoma brucei in culture. Of the other isoprenoids tested, juvenile hormone III and mammalian retinoid X receptor ligands were the most potent trypanocides. Both the procyclic forms and the bloodstream trypomastigotes are killed by these compounds with LD50 values of 5-30 microM. Of the two methoprene stereoisomers, the EE form was the most active, suggesting that a protein target may be involved in mediating effects of these analogues against the parasite. Methoprene was not, however, able to clear trypanosomes from the blood of infected mice. Methoprene acid, the immediate downstream metabolite of methoprene, is not an effective anti-trypanosomal agent, suggesting that in the mice methoprene is converted to an inactive compound. Since methoprene and its analogues have low and well characterized toxicity in mammals these studies stress the importance of further exploring these isoprenoids as lead compounds for the treatment of African sleeping sickness.

Animals↗

PubMed1997

Characterization of Trypanosoma brucei pyridoxal kinase: purification, gene isolation and expression in Escherichia coli.

Pyridoxal kinase catalyzes the ATP-dependent phosphorylation of vitamin B6, generating pyridoxal-5'-phosphate, an important cofactor for many enzymatic reactions. Pyridoxal kinase was purified 4300-fold to homogeneity from Trypanosoma brucei and peptides generated by proteolysis were subjected to amino acid sequence analysis. The peptide sequence information was used to generate a partial clone of T. brucei pyridoxal kinase by polymerase chain reaction (PCR), which in turn was used to screen a T. brucei genomic library for a full length clone. The 903-bp gene was sequenced and found to encode a 300-amino acid protein. The deduced amino acid sequence contains all of the peptide sequences obtained from the proteolytic cleavage of the native enzyme and shares 28% sequence identity with a putative Escherichia coli pyridoxal kinase, identified for its ability to compliment pyridoxal kinase deficient cells. The T. brucei pyridoxal kinase gene was expressed in E. coli and the purified enzyme was found to have pyridoxal kinase activity, confirming that this gene encodes the functional T. brucei enzyme. Native and recombinant pyridoxal kinase have a monomer molecular weight of 37 kDa by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and are dimers in solution. Native T. brucei pyridoxal kinase catalyzes the phosphorylation of pyridoxal with a specific activity of 990 nmol min(-1) per mg and apparent Km values for pyridoxal and ATP of 22 and 9 microM. respectively. Substrate inhibition is observed for pyridoxal. Similar results were obtained for the recombinant enzyme.

Amino Acid Sequence↗

PubMed1997

Arsenate suppression of human keratinocyte programming.

The human keratinocyte line SCC-9 has been used as a model for arsenate-induced perturbations of differentiation. Growth of these cells in 10 microM arsenate permitted the cultures to reach confluence, but prevented expression of 6 markers of suprabasal differentiation (involucrin, loricrin, filaggrin, spr 1, keratin 1 and keratin 10) as assayed by Northern blotting. By contrast, only slight alterations in mRNA levels were observed for one differentiation marker (keratinocyte transglutaminase) and for keratin 5, keratin 14, AP2 or glyceraldehyde phosphate dehydrogenase. The transition metal oxyanions vanadate and chromate had essentially the same suppressive effect on these markers as arsenate, while chronic treatment with tetradecanoylphorbol acetate was generally less effective in suppressing differentiation. To determine whether the previously observed arsenate-mediated alteration in AP1 and AP2 activities could account for the suppression of involucrin, a promoter analysis was conducted. Putative AP1 and AP2 response elements were identified in regions important for transcriptional activity of the 5'-flanking DNA. Mutations in two AP1 sites and one AP2 site were observed to decrease promoter activity significantly, and in combination, to reduce it to approximately 10% of that conferred by the native sequence. These results lend support to the working hypothesis that arsenate suppresses involucrin expression, and, more generally, keratinocyte programming, by altering the transcription factors AP1 and AP2.

Arsenates↗

PubMed1996

Identification of phosphorylation sites in keratinocyte transglutaminase.

Phosphorylation of keratinocyte transglutaminase occurs in its N-terminal extension and is stimulated several-fold by the protein kinase C agonist phorbol myristate acetate. In the present work, this stimulation was prevented by simultaneous treatment of the cells with the protein kinase C-selective inhibitor GF109203X. In contrast, phosphorylation occurring in the absence of phorbol ester was essentially unaffected by GF109203X, although it was decreased dramatically by the non-selective kinase inhibitor staurosporine. Four serine residues that are subject to phosphorylation have been identified by sequencing of radioactive tryptic peptides. Serines at positions 24 and 92, each containing 2-8% of the total radioactivity with or without phorbol ester stimulation, were minor sites of phosphorylation. Serine-82 was by far the dominant site of phosphorylation in the absence of phorbol ester treatment, and was also the major site in its presence. Serine-85 was phosphorylated to a high degree in the presence but not in the absence of phorbol ester stimulation. Thus the data indicate the influence of at least two different kinase activities in transglutaminase phosphorylation.

3T3 Cells↗

PubMed1996

Characterization of Trypanosoma brucei gamma-glutamylcysteine synthetase, an essential enzyme in the biosynthesis of trypanothione (diglutathionylspermidine).

The parasitic protozoan Trypanosoma brucei maintains redox balance by synthesizing a conjugate of glutathione and spermidine termed trypanothione. The first committed step in the biosynthesis of glutathione, and thereby trypanothione, is catalyzed by the enzyme gamma-glutamylcysteine synthetase (gammaGCS). We have cloned and sequenced the 2037-base pair gene coding for the catalytic subunit of T. brucei gammaGCS. T. brucei gammaGCS appears to be encoded by a single copy gene. A transcript of about 2.3 kilobases was observed in procyclic trypomastigotes. The deduced amino acid sequence of 679 amino acids shares 45, 41, and 36% sequence identity with mammalian, Caenorhabditis elegans, and yeast gammaGCS, respectively. The T. brucei gammaGCS gene was expressed in E. coli; the purified 77.4-kDa enzyme catalyzes the ligation of L-Glu to L-Cys with a kcat of 10 s-1, confirming that the gene encodes the functional catalytic subunit of gammaGCS. The apparent Km values measured for the three natural substrates L-Glu, L-Cys, and ATP are 0.24, 0.69, and 0.07 mM, respectively. Unlike the mammalian enzyme, L-alpha-aminobutyrate (apparent Km = 10 mM) is a poor substitute for L-Cys in the T. brucei gammaGCS-catalyzed reaction. T. brucei gammaGCS is feedback-inhibited by glutathione (apparent KI = 1.1 mM), and it is inactivated by cystamine and buthionine sulfoximine. The kinetic properties of recombinant T. brucei gammaGCS suggest that the substrate binding pocket and the mechanism of enzyme regulation differ from the mammalian enzyme, providing evidence that T. brucei gammaGCS could be a selective chemotherapeutic target for the treatment of trypanosomiasis.

Amino Acid Sequence↗

PubMed1996

Circular dichroism assay for decarboxylation of optically pure amino acids: application to ornithine decarboxylase.

A circular dichroism (CD) assay for decarboxylation of optically pure amino acids is described. The viability of this assay is demonstrated using the Trypanosoma brucei ornithine decarboxylase (ODC)-catalyzed reaction of L-ornithine to putrescine and CO2. The results from the CD assay (kcat of 7.5 +/- 0.7 s-1 and Km 230 +/- 60 microM) were identical to the results obtained from the commercially available dye-linked assay which couples CO2 production with NADH oxidation (kcat of 7.3 +/- 0.5 s-1 and Km 320 +/- 30 microM). The CD assay has advantages over the currently used 14CO2 and dye-linked assays since it can be continuously monitored and does not contain additional enzymes. The CD assay will enable the determination of the effects of pH, ionic strength, and D2O on catalysis by ODC. Furthermore, the availability of cuvets with pathlengths from 0.01 to 100 mm provides an effective range for the CD assay from 10 microM to 2.5 M L-ornithine concentration for this assay. This technique should be generally applicable for steady-state analysis of other decarboxylases but is not easily amenable to the analysis of crude enzyme preparations.

Cell Line↗

PubMed1996

Role of the prodomain in folding and secretion of rat pancreatic carboxypeptidase A1.

Pancreatic carboxypeptidase A1 (CPA1) is synthesized as an inactive precursor, proCPA1, which is processed to the active enzyme by the proteolytic removal of the 95-amino acid N-terminal prodomain. Purified rat proCPA1 is renatured in vitro after denaturation in guanidine or in guanidine plus reducing agents. In contrast, purified CPA1 is not renatured under any of the conditions tested. While proCPA1 is secreted in yeast when fused to the alpha-factor signal sequence in place of its endogenous signal sequence, mature CPA1 is not secreted and is trapped and degraded intracellularly. Thus, in addition to maintaining CPA1 in the inactive state, the prodomain promotes folding and secretion of the proenzyme. Neither of these functions can be restored by supplying the prodomain to CPA1 in trans. The three-dimensional structure of porcine proCPA reveals a number of extensive contacts made between the prodomain and the enzyme active site which account for its inhibitory properties [Guasch et al. (1992) J. Mol. Biol. 224, 141-157]. Among these contacts are salt bridges formed between Arg-71 and Asp-A36 and between Arg-124 and Asp-A89. Mutation of any of these four residues inhibits secretion of proCPA1 from yeast and results in its intracellular degradation. The effect of the mutations on secretion suggests that interactions which stabilize the binding of prodomain to the native enzyme active site may also be important for the successful folding of proCPA1.

Amino Acid Sequence↗

PubMed1996

Crystallization and preliminary X-ray studies of ornithine decarboxylase from Trypanosoma brucei.

Trypanosoma brucei ornithine decarboxylase, expressed and purified from E. coli, has been crystallized by the vapor diffusion method using PEG 3350 as a precipitant. The crystals belong to the monoclinic space group P2(1) and have cell constants of a = 66.3 angstroms, b = 151.8 angstroms, c = 83.7 angstroms, and beta = 101.2 degrees. While larger crystals are twinned, smaller crystals (0.4 x 0.3 x 0.05 mm3) are single.

Animals↗

PubMed1996

Alteration by 2,3,7,8-Tetrachlorodibenzo-p-dioxin of CCAAT/enhancer binding protein correlates with suppression of adipocyte differentiation in 3T3-L1 cells.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related compounds elicit multiple effects on the function of adipose tissue and adipogenic cell lines, including the suppression of adipocyte differentiation. We began to examine the mechanism by which TCDD inhibits differentiation of the established preadipocyte cell line 3T3-L1. Examination of the expression of several early marker genes of preadipocyte differentiation through Northern blot analysis and of differentiation-dependent mitosis showed that TCDD did not interfere with the earliest known responses of preadipocytes to inducers of differentiation. Analysis of mRNA for three isoforms of the CCAAT/enhancer binding protein (C/EBP) revealed that TCDD (5 nM) selectively inhibited the induction of C/EBP alpha mRNA but did not block the induction of C/EBP beta and C/EBP delta in response to differentiation inducers. The differentiation-dependent induction of PPAR gamma mRNA was also blocked by TCDD. Immunoblot analysis with specific antibodies to each C/EBP isoform demonstrated that the levels of C/EBP delta and C/EBP beta protein were rapidly induced (by day 1) and then abrogated by day 4 and 8, respectively, in solvent-treated (control) cells. In TCDD-treated cells, however, the levels of C/EBP beta and C/EBP delta protein persisted at these time points. In contrast, C/EBP alpha protein was markedly suppressed by TCDD in concordance with its level of RNA. Both translational products of C/EBP alpha, p30 and p42, were dose-dependently decreased by TCDD. Gel shift analysis of nuclear extract binding to an oligonucleotide containing a C/EBP DNA recognition sequence revealed no difference between extracts from control and TCDD-treated cells in the binding pattern at day 2 of differentiation. At days 4 and 8, the band corresponding to the C/EBP alpha/DNA complex (as determined with supershift assays) was dramatically decreased in the treated extracts in comparison to control extracts. In contrast, a band corresponding to a C/EBP beta/DNA complex was found to be enriched in the treated samples. These data indicate that suppression of differentiation in the 3T3-L1 preadipocyte cell line by TCDD occurs at a short but defined period, during the differentiation program, and involves altered regulation of C/EBP, including the inhibition of C/EBP alpha.

3T3 Cells↗

PubMed1995

Domain organization and a protease-sensitive loop in eukaryotic ornithine decarboxylase.

Trypanosoma brucei ornithine decarboxylase was reconstituted by coexpression of two polypeptides corresponding to residues 1-305 and residues 306-425 in Escherichia coli. The two peptides were coexpressed, at wild-type levels, from a single transcriptional unit that was separated by a 15-nucleotide untranslated region containing a ribosome binding site. The fragmented enzyme was purified and analyzed. The N- and C-terminal peptides are tightly associated into a fully active tetramer which has the same molecular weight as the native dimer. The kinetic constants (Km and kcat) measured for the decarboxylation of ornithine are identical to those obtained for the wild-type enzyme. These results suggest that the enzyme is organized into two structural domains, with a domain boundary in the region of amino acid 305. In contrast, the individual N- and C-terminal peptides are expressed primarily as inclusion bodies. Small quantities of soluble N-terminal peptide could be purified. This truncated protein is capable of inhibiting the wild-type enzyme, suggesting that it is folded into a native-like structure. Limited proteolysis with trypsin or chymotrypsin identifies a likely surface loop at amino acids 160-170, present in both the mouse and T. brucei enzyme, which positions one or more functionally important active site residues (e.g., Lys169). Kinetic analysis of a chimeric enzyme composed of T. brucei and mouse ornithine decarboxylase suggests that the substrate carboxylate binding determinant is located between residues 1 and 170.

Amino Acid Sequence↗

PubMed1995

Acidic residues important for substrate binding and cofactor reactivity in eukaryotic ornithine decarboxylase identified by alanine scanning mutagenesis.

Ornithine decarboxylases from Trypanosoma brucei, mouse, and Leishmania donovani share strict specificity for three basic amino acids, ornithine, lysine, and arginine. To identify residues involved in this substrate specificity and/or in the reaction chemistry, six conserved acidic resides (Asp-88, Glu-94, Asp-233, Glu-274, Asp-361, and Asp-364) were mutated to alanine in the T. brucei enzyme. Each mutation causes a substantial loss in enzyme efficiency. Most notably, mutation of Asp-361 increases the Km for ornithine by 2000-fold, with little effect on kcat, suggesting that this residue is an important substrate binding determinant. Mutation of the only strictly conserved acidic residue, Glu-274, decreases kcat 50-fold; however, substitution of N-methylpyridoxal-5'-phosphate for pyridoxal-5'-phosphate as the cofactor in the reaction restores the kcat of E274A to wild-type levels. These data demonstrate that Glu-274 interacts with the protonated pyridine nitrogen of the cofactor to enhance the electron withdrawing capability of the ring, analogous to Asp-222 in aspartate aminotransferase (Onuffer, J. J., and Kirsch, J. F. (1994) Protein Eng. 7, 413-424). Eukaryotic ornithine decarboxylase is a homodimer with two shared active sites. Residues 88, 94, 233, and 274 are contributed to each active site from the same subunit as Lys-69, while residues 361 and 364 are part of the Cys-360 subunit.

Animals↗