Altering the reaction specificity of eukaryotic ornithine decarboxylase.

Ornithine decarboxylase (ODC) catalyzes the first committed step in the biosynthesis of polyamines, and it has been identified as a drug target for the treatment of African sleeping sickness, caused by Trypanosoma brucei. ODC is a pyridoxal 5'-phosphate (PLP) dependent enzyme and an obligate homodimer. X-ray structural analysis of the complex of the T. brucei wild-type enzyme with the product putrescine reveals two structural changes that occur upon ligand binding: Lys-69 is displaced by putrescine and forms new interactions with Glu-94 and Asp-88, and the side chain of Cys-360 rotates into the active site to within 3.4 A of the imine bond. Mutation of Cys-360 to Ala or Ser reduces the k(cat) of the decarboxylation reaction by 50- and 1000-fold, respectively. However, HPLC analysis of the products demonstrates that the mutant enzymes almost exclusively catalyze a decarboxylation-dependent transamination reaction to form pyridoxamine 5-phosphate (PMP) and gamma-aminobutyraldehyde, instead of PLP and putrescine. This side reaction arises when the decarboxylated substrate intermediate is protonated at C4' of PLP instead of at the C(alpha) of substrate. For the reaction catalyzed by the wild-type enzyme, this side reaction occurs infrequently (<0.01% of the turnovers). Single turnover analysis and multiwavelength stopped-flow spectroscopic studies suggest that for the mutant ODCs protonation at C4' occurs either very rapidly or in a concerted reaction with decarboxylation and that the rate-limiting step in the steady-state reaction is Schiff base hydrolysis/product release. These studies demonstrate a role for Cys-360 in the control of the C(alpha) protonation step that catalyzes the formation of the physiological product putrescine. The results further provide insight into the mechanism by which this class of PLP-dependent enzymes controls reaction specificity.

Treasure of the Past: IV: THE STANDARD-CELL COMPARATOR, A SPECIALIZED POTENTIOMETER.

The standard cell plays a very important role in the maintenance of the electrical units and in correlating the units of the various national laboratories. Modern standard cells have attained such a high degree of reproducibility and permanence as to warrant the use of apparatus of the utmost precision and reliability in their intercomparison. The paper describes a new potentiometer developed especially for this purpose. Although it actually measures the small difference between the known emf of a reference cell and that of the cell under test, it contains a simple mechanical computing feature which automatically adds this small difference algebraically to the emf of the reference cell and thereby indicates directly the value of the emf under measurement. The design of the instrument is such that no readjustment of its coils will be required when the impending changes in the ohm and the volt are accomplished. The new instrument has been given the distinctive name, "standard-cell comparator".

X-ray structure of ornithine decarboxylase from Trypanosoma brucei: the native structure and the structure in complex with alpha-difluoromethylornithine.

Ornithine decarboxylase (ODC) is a pyridoxal 5'-phosphate (PLP) dependent homodimeric enzyme. It is a recognized drug target against African sleeping sickness, caused by Trypanosoma brucei. One of the currently used drugs, alpha-difluoromethylornithine (DFMO), is a suicide inhibitor of ODC. The structure of the T. brucei ODC (TbODC) mutant K69A bound to DFMO has been determined by X-ray crystallography to 2.0 A resolution. The protein crystallizes in the space group P2(1) (a = 66.8 A, b = 154.5 A, c = 77.1 A, beta = 90.58 degrees ), with two dimers per asymmetric unit. The initial phasing was done by molecular replacement with the mouse ODC structure. The structure of wild-type uncomplexed TbODC was also determined to 2.9 A resolution by molecular replacement using the TbODC DFMO-bound structure as the search model. The N-terminal domain of ODC is a beta/alpha-barrel, and the C-terminal domain of ODC is a modified Greek key beta-barrel. In comparison to structurally related alanine racemase, the two domains are rotated 27 degrees relative to each other. In addition, two of the beta-strands in the C-terminal domain have exchanged positions in order to maintain the location of essential active site residues in the context of the domain rotation. In ODC, the contacts in the dimer interface are formed primarily by the C-terminal domains, which interact through six aromatic rings that form stacking interactions across the domain boundary. The PLP binding site is formed by the C-termini of beta-strands and loops in the beta/alpha-barrel. In the native structure Lys69 forms a Schiff base with PLP. In both structures, the phosphate of PLP is bound between the seventh and eighth strands forming interactions with Arg277 and a Gly loop (residues 235-237). The pyridine nitrogen of PLP interacts with Glu274. DFMO forms a Schiff base with PLP and is covalently attached to Cys360. It is bound at the dimer interface and the delta-carbon amino group of DFMO is positioned between Asp361 of one subunit and Asp332 of the other. In comparison to the wild-type uncomplexed structure, Cys-360 has rotated 145 degrees toward the active site in the DFMO-bound structure. No domain, subunit rotations, or other significant structural changes are observed upon ligand binding. The structure offers insight into the enzyme mechanism by providing details of the enzyme/inhibitor binding site and allows for a detailed comparison between the enzymes from the host and parasite which will aid in selective inhibitor design.

Lysine-69 plays a key role in catalysis by ornithine decarboxylase through acceleration of the Schiff base formation, decarboxylation, and product release steps.

Ornithine decarboxylase (ODC) is a pyridoxal-5'-phosphate-dependent (PLP) enzyme that catalyzes the biosynthesis of the polyamine putrescine. Similar to other PLP-dependent enzymes, an active site Lys residue forms a Schiff base with PLP in the absence of substrate. The mechanistic role of this residue (Lys-69) in catalysis by Trypanosoma brucei ODC has been studied by analysis of the mutant enzymes, in which Lys-69 has been replaced by Arg (K69R ODC) and Ala (K69A ODC). Analysis of K69A ODC demonstrated that the enzyme copurified with amines (e.g. putrescine) that were tightly bound to the active site through a Schiff base with PLP. In contrast, on the basis of an absorption spectrum of K69R ODC, PLP is likely to be bound to this mutant enzyme in the aldehyde form. Pre-steady-state kinetic analysis of the reaction of K69R ODC with L-Orn and putrescine demonstrated that the rates of both the product release (k(off.Put) = 0.0041 s(-)(1)) and the decarboxylation (k(decarb) = 0.016 s(-)(1)) steps were decreased by10(4)-fold in comparison to wild-type ODC. Further, the rates of Schiff base formation between K69R ODC and either substrate or product have decreased by at least 10(3)-fold. Product release remains as the dominant rate-limiting step in the reaction (the steady-state parameters for K69R ODC are k(cat) = 0.0031 s(-)(1) and K(m) = 0.18 mM). The effect of mutating Lys-69 on the decarboxylation step suggests that Lys-69 may play a role in the proper positioning of the alpha-carboxylate for efficient decarboxylation. K69R ODC binds diamines and amino acids with higher affinity than the wild-type enzyme; however, Lys-69 does not mediate substrate specificity. Wild-type and K69R ODC have similar ligand specificity preferring to bind putrescine over longer and shorter diamines. Kinetic analysis of the binding of a series of diamines and amino acids to K69R ODC suggests that noncovalent interactions in the active site of K69R ODC promote selective ligand binding during Schiff base formation.

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.

Five hundred consecutive carotid endarterectomies: emphasis on vein patch closure.

This study reviews 500 consecutive carotid endarterectomies performed in 429 patients in one practice over a 12-year period, emphasis being placed on the technique of vein patch closure and its durability. The records of all such patients were reviewed. Data collected included indication, age, sex, angiogram and duplex scan results, technique of carotid closure, complications within 30 days, and follow-up postoperative duplex scans. The technique emphasized generous exposure, distal arteriotomy, routine shunting, and narrow vein patch angioplasty. The mean patient age was 68 years; 245 (57.1%) were men, and 184 (42.9%) were women. Indications for surgery were transient ischemic attack 256 (51.2%); symptom-free stenosis 144 (28.8%); recovered stroke 60 (12%); and non-hemispheric symptoms 40 (8%). The arteriotomy was closed primarily in 71 (14.2%) instances and with a patch in 429 (85.8%). Complications included five (1%) deaths, one (0.2%) stroke, nine (1.8%) transient ischemic attacks, and four (0.8%) wound hematomas. One (0.2%) vein patch rupture occurred. Serial postoperative duplex scans were reviewed in 455 (91%) patients. No significant residual disease was found in any of these patients; three (0.7%) patients were identified with recurrent symptom-free stenoses of >80%; one (0.2%) silent carotid occlusion occurred; and no aneurysms were identified. Classic descriptions of carotid endarterectomy limited the carotid arteriotomy to the bulb area, while contemporary carotid surgery emphasizes wide internal carotid exposure and distal arteriotomy. The authors' experience with vein patch closure confirms the validity of this technique and its low short- and long-term morbidity.

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.

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.

Evidence for a tryptophan tryptophylquinone aminosemiquinone intermediate in the physiologic reaction between methylamine dehydrogenase and amicyanin.

The tryptophan tryptophylquinone (TTQ) cofactor of methylamine dehydrogenase (MADH) is covalently modified by nitrogen during its two-electron reduction by methylamine to form an aminoquinol (N-quinol). It is possible, in vitro, to generate unmodified O-quinol and O-semiquinone forms of MADH with dithionite, as well as an N-semiquinone form which contains a substrate-derived nitrogen. Rapid-scanning stopped-flow spectroscopy and global kinetic analysis are used to demonstrate that N-semiquinone is a true physiologic reaction intermediate which accumulates during the two sequential one-electron oxidations of N-quinol MADH by amicyanin. In contrast, no detectable O-semiquinone accumulates during the two sequential one-electron oxidations of the O-quinol form of MADH by amicyanin. This is because the reaction of N-semiquinone with amicyanin is much slower (70 s-1 at 25 degrees C) than the reaction of O-semiquinone ( > 1000 s-1). These rate constants obtained from global analysis of the overall reaction are the same as those obtained when each semiquinone form was made in vitro and then mixed with oxidized amicyanin. The presence of 200 mM NH4Cl during the reaction of O-quinol MADH with amicyanin does not cause any detectable accumulation of a semiquinone species. Thus, the accumulation of the intermediate in the reactions of the N-quinol is not due to the influence of noncovalently bound ammonia at the active site of the O-semiquinone. These data indicate that the intermediate which accumulates during the complete oxidation of substrate-reduced N-quinol MADH is not the O-semiquinone, but the more slowly reacting N-semiquinone, and that the N-semiquinone is a physiologically relevant reaction intermediate. These results also provide good evidence in favor of an aminotransferase mechanism, as opposed to an imine elimination mechanism, for the reaction of MADH with substrate methylamine.

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.

Kinetic and thermodynamic analysis of a physiologic intermolecular electron-transfer reaction between methylamine dehydrogenase and amicyanin.

The quinoprotein methylamine dehydrogenase (MADH) and a type I copper protein, amicyanin, form a physiologic complex in which electrons are transferred from tryptophan tryptophylquinone to copper. The reoxidation of MADH by amicyanin has been studied by stopped-flow spectroscopy. The rate constant for the electron-transfer (ET) reaction and the dissociation constant for the complex have been determined at different temperatures. Marcus theory was used to calculate the distance, reorganizational energy, and electronic coupling for the intermolecular ET reaction. The ET reaction exhibited a large apparent reorganizational energy of approximately 225 kJ mol-1 (2.3 eV) and a coupling of approximately 11.7 cm-1. From X-ray crystallographic studies of an actual complex of these proteins from Paracoccus denitrificans [Chen, L., et al. (1992) Biochemistry 31, 4959-4964], it was possible to infer putative pathways of ET. The ET distance predicted by Marcus theory from kinetic data correlated reasonably well with the structural information. Thus, it has been possible to correlate ET theories with data from solution studies and a known structure for a naturally occurring ET reaction between soluble proteins.

Perception of quality of life before and after disclosure of trial results: a report from the Program on the Surgical Control of the Hyperlipidemias (POSCH).

The Program on the Surgical Control of the Hyperlipidemias (POSCH) was a randomized controlled clinical trial designed to ascertain whether cholesterol lowering induced by the partial ileal bypass operation would favorably affect overall mortality and the mortality and morbidity due to coronary heart disease. The trial results provided strong clinical and coronary arteriographic support for the beneficial effects of lipid modification for the reduction of atherosclerosis progression. At the same time, the surgery-assigned group experienced diarrhea and an increased incidence of kidney stones and gallstones compared to the control-assigned group. Identical quality of life determinations were performed in the POSCH study population shortly before disclosure of the trial results to the patients and shortly thereafter. The purpose of this dual assessment was to evaluate the effect of knowledge of outcomes on the patients' subjective evaluation of quality of life. The primary instrument utilized for analysis of the perception of quality of life in POSCH was the McMasters Health Index Questionnaire (MHIQ). In addition, four study-specific questions were asked of the trial patients. The results for the MHIQ before disclosure of trial results showed a difference (p = 0.07) favoring the control-assigned group (diet-treated), for the social function index of the MHIQ. After disclosure of the trial results, the difference was larger (p < 0.05). For the four study-specific questions, all differences favored the control-assigned group (p < 0.01) before and after disclosure of the trial results, with the exception of satisfaction with randomization allocation in the surgery-assigned group (p = 0.08). The intragroup MHIQ indices before and after disclosure of the trial results showed no suggestive significant differences, except in the surgery-assigned group, in which there was an improvement in the emotional function index after disclosure of the trial results (p = 0.03). The intragroup responses to the study-specific questions before and after disclosure of the trial results again showed no significant differences, except in the surgery-assigned group, in which there was an improvement in patient satisfaction with randomization allocation after disclosure of the trial results (p = 0.04).(ABSTRACT TRUNCATED AT 400 WORDS)

A method for extracting rate constants from initial rates of stopped-flow kinetic data: application to a physiological electron-transfer reaction.

The most commonly used methods for analysis of stopped-flow kinetic data require performing a series of measurements in which one reactant is varied at concentrations significantly greater than the concentration of the other reactant. For enzyme-catalysed reactions this may not be possible, because the dissociation constants for the enzyme-substrate complex are often of the same order of magnitude as the high concentrations of enzyme that must frequently be used in stopped-flow studies. An alternative method of data analysis is presented which allows the determination of microscopic rate constants from initial rates of stopped-flow kinetic data in which substrate is varied in a range of concentrations approximately the same as the enzyme. This method also provides a simple and accurate method for determining k4, the rate of the reverse reaction. This method has been used to describe a physiological electron transfer reaction between a quinoprotein, methylamine dehydrogenase, and a copper protein, amicyanin. At 20 degrees C, the rate of the electron-transfer reaction from methylamine dehydrogenase to amicyanin was 24 s-1, and the dissociation constant for complex-formation was 1.9 microM.

Deuterium kinetic isotope effect and stopped-flow kinetic studies of the quinoprotein methylamine dehydrogenase.

Stopped-flow kinetic studies of the reductive half-reaction of methylamine dehydrogenase from Paracoccus denitrificans yielded kinetic constants for the reversible formation of the imine intermediate formed between the substrate and the tryptophan tryptophylquinone (TTQ) prosthetic group and for the hydrogen abstraction step which occurs concomitantly with TTQ reduction. When CD3NH2 was used as a substrate, deuterium kinetic isotope effects of 4.2 and 3.8, respectively, were measured for the rate constants that correspond to the formation and dissociation of the iminoquinone intermediate. A deuterium kinetic isotope effect of 17.2 was measured for the hydrogen abstraction step. The maximum deuterium kinetic isotope effect which was measured in steady-state kinetic experiments was 3.0. These data are discussed in relation to the reaction mechanism of methylamine dehydrogenase and the similar large deuterium kinetic isotope effect for hydrogen abstraction which has been observed for another quinoprotein, plasma amine oxidase.

Stabilization of thermally labile alkylcobalamins by a haptocorrin from chicken serum.

Binding of neopentylcobalamin and benzylcobalamin to the apoprotein of a haptocorrin from chicken serum has been demonstrated spectrophotometrically. The spectra of the protein-bound cobalamins strongly resemble those of base-on alkylcobalamins and show that when unbound these sterically hindered alkylcobalamins are only approximately 75% (benzyl) and 40% (neopentyl) base-on, at neutral pH and at 5 degrees C. The haptocorrin was found to stabilize the spontaneous thermal decomposition of the neutral species of benzylcobalamin and neopentylcobalamin by 470-fold (3.6 kcal) and 166-fold (3.0 kcal), respectively, relative to the protein-free species. After correction of the activation parameters for the thermal decomposition of the protein-free, neutral alkylcobalamins for the relative proportions of base-on and base-off species, the haptocorrin was found to stabilize the base-on species of both alkylcobalamins by 275- to 1400-fold (approximately 3.3 to 4.3 kcal). From the temperature dependence of the decomposition reactions, the enthalpies of activation are found to be essentially identical for the protein-free and protein-bound species of either cobalamin. Thus, stabilization of the thermal decomposition of these sterically hindered alkylcobalamins by haptocorrin is entirely due to entropic factors.

Determination of fluorocarbon in blood.

Because fluorocarbons can dissolve relatively large quantities of oxygen and carbon dioxide, there is considerable interest in utilizing them to develop new methods of extracorporael circulation, artificial red blood cells, and liquid breathing techniques. A method for the assay of fluorocarbon in blood is presented. The fluorocarbon is extracted from the blood with toluene, and fluoride is released from the fluorocarbon in the toluene extract by reaction with sodium biphenyl. The inorganic fluoride is then extracted with aqueous sodium acetate, the pH of the extract is adjusted, and the activity of the fluoride ion is read with a fluoride-specific ion electrode. The assay was effective for fluorocarbon concentrations in the range of 1 to 30 ppm.