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PubMed2006

Mechanistic applicability domains for nonanimal-based prediction of toxicological end points: general principles and application to reactive toxicity.

In light of new legislation (e.g., the REACH program in the European Union), several initiatives have recently emerged to increase acceptance of (quantitative) structure-activity relationships [(Q)SARs] to reduce reliance on animal (in vivo) testing. Among the principles for assessing the validity of (Q)SARs is the need for a defined domain of applicability, i.e., identification of the range of compounds for which the (Q)SAR can confidently be applied for purposes of toxicity prediction. Here, we attempt to develop a "natural" classification into applicability domains based on considering how a compound and the target organism between them "decide" on the nature and extent of the toxic effect. With particular emphasis on reactive toxicity, we present rules, based on organic reaction mechanistic principles, for classifying reactive toxicants into their appropriate mechanistic applicability domains.

Animal Testing Alternatives↗

PubMed1996

Hydrotropic solubilization--mechanistic studies.

PURPOSE: This study examines the mechanism of hydrotropic solubilization using the riboflavin-nicotinamide system. The most commonly proposed mechanism for hydrotropic solubilization is complexation, and therefore, is investigated. Additionally, since nicotinamide and several other hydrotropic agents self-associate in aqueous solution, the possibility that self-association of the hydrotropic agent is important mechanistically is examined by studying the effect of temperature on hydrotropic ability. Researchers have shown that the degree of self association decreases with increasing temperature. Therefore, if temperature affects the solubilizing capacity of nicotinamide, self-association must be mechanistically significant. METHODS: The complexation hypothesis is tested by looking at nicotinamide's ability to quench riboflavin fluorescence and by examining changes in the UV/Vis spectrum of riboflavin upon addition of nicotinamide. The solubility of riboflavin in nicotinamide solutions as a function of temperature is determined to assess the impact of self-association on hydrotropicity. RESULTS: Nicotinamide does not alter the intrinsic fluorescence of riboflavin nor are changes indicative of complexation observed in the UV/Vis spectrum Temperature does have an effect on the hydrotropic ability of nicotinamide. Specifically, as temperature increases, the solubilizing capacity of nicotinamide decreases. CONCLUSIONS: Because nicotinamide is unable to quench riboflavin fluorescence, and does not produce significant spectral changes, complexation of hicotinamide and riboflavin does not occur. However, since increasing temperature causes a decrease in the hydrotropic ability of nicotinamide and in its degree of self-association, it is proposed here that the self-association of nicotinamide impacts the hydrotropic mechanism.

Chemistry, Physical↗

PubMed1998

Quantitative structure-pharmacokinetics relationships: II. A mechanistically based model to evaluate the relationship between tissue distribution parameters and compound lipophilicity.

The tissue-to-unbound plasma distribution coefficients (Kpus) of 14 rat tissues after i.v. administration of nine 5-n-alkyl-5-ethyl barbituric acids, determined in a previous study, were used to identify a model of the relationship between tissue distribution and lipophilicity of the homologs, expressed in terms of their octanol to water partition ratio, P. Based on mechanistic considerations and assumptions, the parameter model was expressed as Kpu tau = fw.tau [1 + a tau (nPt.tau)Pb tau], where fw.tau is the tissue water content. (nPt. tau) is the binding capacity of the tissue, n is the number of the binding sites, a tau and b tau are the parameters of the relationship Ka tau = a tau Pb tau; and Ka tau is the binding association constant of each tissue. The parameter model was linearized and fitted to the predetermined Kpu values, yielding correlation coefficients ranging between .940 and .997. The predictive performance of the parameter model was evaluated using a leave-one-out procedure with subsequent computation of the mean prediction error (ME = measurement of the prediction bias) and the square root of the mean squared prediction error (RMSE = measurement of the prediction accuracy). The ME varied between -22.48 and 61.14%, indicating a slight tendency for overpredicting. The RMSE was between 24.73 and 102% for the individual tissues across the different homologs; and between 28.33 and 85.2% for the individual homologs across the different tissues. The apparently high Kpu prediction errors, when translated through the low sensitivity of the barbiturate whole-body physiologically based pharmacokinetic model, established previously, leads to predicted tissue concentration-time profiles within 5 to 20% of the original ones. Therefore, it is concluded, that the identified mechanistically based model is a good predictor of the tissue-to-unbound Kpus in the rat tissues.

Animals↗

PubMed1992

Radical behaviorism and scientific frameworks. From mechanistic to relational accounts.

A substantial portion of B. F. Skinner's scholarship was devoted to developing methods and terms for a scientific study of behavior. Three concepts central to scientific accounts--cause, explanation, and theory--are examined to illustrate the distinction between mechanistic and relational frameworks and radical behaviorism's relationship to those frameworks. Informed by a scientific tradition that explicitly rejects mechanistic interpretations, radical behaviorism provides a distinctive stance in contemporary psychology. The present analysis suggests that radical behaviorism makes closer contact with the "new world view" advocated by physicists and philosophers of science than does much of contemporary psychology.

Behaviorism↗

PubMed1992

From mechanistic to functional behaviorism.

A shift from mechanistic behaviorism to functional behaviorism is presented against the background of two historical traditions, one with an emphasis on form, the other with an emphasis on function. Skinner's work, which made more contributions to a functional behaviorism than to a mechanistic behaviorism, exemplifies this shift. The two traditions and an account of Skinner's development of functional relations are presented in order to show Skinner's contributions to aligning modern behavior analysis with the functional tradition.

Behaviorism↗

PubMed1990

Mandelate racemase and muconate lactonizing enzyme are mechanistically distinct and structurally homologous.

Mandelate racemase (MR) and muconate lactonizing enzyme (MLE) catalyse separate and mechanistically distinct reactions necessary for the catabolism of aromatic acids by Pseudomonas putida. The X-ray crystal structure of MR, solved at 2.5 A resolution, reveals that the secondary, tertiary and quaternary structures of MR and MLE are remarkably similar; also, MR and MLE are about 26% identical in primary structure. However, MR has no detectable MLE activity and vice versa. Thus, MR and MLE constitute the first example of enzymes that catalyse different reactions, as opposed to mechanistically identical reactions on different substrates, yet possess sufficient structural and sequence identity that they are likely to have evolved from a common ancestor. The discovery that MR and MLE catalyse different reactions but share a common structural framework has broad implications for the natural evolution of enzymes and metabolic pathways, as well as for the rational modification of enzyme activities.

Binding Sites↗

PubMed1998

Mechanistic and quantitative prediction of aminopeptidase activity in stripped human skin based on the HaCaT cell sheet model.

HaCaT cell culture sheets were recently demonstrated to be a useful tool to study epidermal metabolism. Here we report on a mechanistic and quantitative correlation between the kinetics of aminopeptidase-based cleavage of L-Ala-4-methoxy-2-naphthylamide (Ala-MNA) in HaCaT sheets versus stripped human skin. Fresh human skin (breast or abdominal) was obtained from cosmetic surgery, tape-stripped, and dermatomed. HaCaT sheets were cultured on porous membranes. Diffusion and concurrent metabolism were studied under reflection and permeation conditions. Numerical simulations of simultaneous diffusion and saturable Michaelis-Menten metabolism were based on a physical model and a fixed set of independently obtained parameters (diffusion coefficient D, distance x, partition coefficient P, Michaelis constant Km, maximum metabolic rate Vmax). Under reflection conditions, cleavage of Ala-MNA in HaCaT sheets was very close to stripped skin. In contrast, in permeation studies substrate only permeated through HaCaT whereas passage through stripped skin led to full cleavage of Ala-MNA to MNA. All experimental data were in reasonable to excellent agreement with numerically generated data. Differences between HaCaT and stripped skin could be quantitatively and mechanistically explained by the thickness of the metabolically active layer, i.e., approximately 10 microm in HaCaT and approximately 40 microm in stripped skin. Full cleavage of permeating Ala-MNA in stripped skin was predicted to occur within the upper approximately 20 microm of viable epidermis. Thus epidermal aminopeptidase activity may act as an efficient metabolic barrier to fully block the permeation of aminopeptidase labile xenobiotics. Within the settings of this study the kinetics of metabolism in the viable epidermis of skin is predictable from HaCaT sheets.

2-Naphthylamine↗

PubMed1985

A mechanistic analysis of the National Cooperative Dialysis Study (NCDS).

The purpose of the NCDS was to determine the probability of clinical failure (PF) as a function of the level of dialysis and protein catabolic rate (pcr, g/kg/day). The level of dialysis prescribed in the NCDS was mechanistically defined as Kt/V (product of dialyzer urea clearance and treatment time divided by body urea volume), which exponentially determines decrease in BUN during dialysis and is also a mathematical analogue of pcr, BUN. Mechanistic analysis (MA) showed that PF was a discontinuous function of Kt/V as it was prescribed in the NCDS and that a dependence of PF on pcr could not be assessed because of the study design. The MA results were compared to those reported with statistical analysis (SA) that used BUN and pcr. The SA predicts PF is strongly dependent on pcr with nutrition-dependent high PF for pcr less than or equal to 0.8 and low PF with high pcr and intensive dialysis. The MA suggests SA results may not be valid because a continuous outcome function is assumed and, due to study design, Kt/V was a dependent variable of pcr and these two variables cannot be clearly separated by analysis of BUN and pcr alone.

Blood Urea Nitrogen↗

PubMed2026

Proteomic analysis of pancreatic endocrine cells by mechanistic single-cell isolation identifies membrane pathways.

To better understand diabetes and normoglycemia, pancreatic islet biology requires a precise molecular understanding of islet cell types at both the transcriptomic and proteomic levels. While transcriptomic analyses are well established, comprehensive proteomic characterization has been lacking, limiting our knowledge of islet molecular complexity. Here we introduce a nonenzymatic, mechanistic single-cell isolation technology using laser microdissection (LMD7), facilitating proteomic and transcriptomic analysis of physically isolated α-, β- and δ-cells from fresh-frozen, unfixed pancreatic tissue. This mechanistic approach avoids enzymatic digestion and chemical fixation, preserving the cells' native molecular state before processing. Given the limited existing proteomic data, we supplemented our findings with transcriptomic analysis generated using the same method and compared our results with data from enzymatically isolated cells, obtained by fluorescence-activated cell sorting and compiled by others. Our analysis revealed that enzymatic digestion alters gene expression patterns, particularly those of membrane-associated proteins, underscoring the impact of isolation techniques on biological outcomes. We identified cell-type-specific proteins typically underrepresented in pancreatic single-cell transcriptomic datasets. β-cells exhibited enrichment in vesicle trafficking proteins, α-cells displayed distinct calcium-dependent action potential machinery and δ-cells showed elevated expression of focal adhesion-related proteins. In addition, we report an inverse molecular relationship between β- and δ-cells, potentially driven by transcriptional regulators such as Mlxipl. By establishing robust molecular profiles directly from intact pancreatic tissue, this work provides a reference point for future pathological comparisons, offering a framework to investigate how diabetes and other endocrine disorders reshape islet cell biology.

Journal Article↗

PubMed1997

Profile of SB-204269, a mechanistically novel anticonvulsant drug, in rat models of focal and generalized epileptic seizures.

1. Earlier optimization of structure-activity relationships in a novel series of 4-(benzoylamino)-benzopyrans, led to the discovery of SB-204269 (trans-(+)-6-acetyl-4S-(4-fluorobenzoylamino)-3,4-dihydro-2, 2-dimethyl-2H-benzo[b]pyran-3R-ol, hemihydrate), a potent orally-active anticonvulsant in the mouse maximal electroshock seizure threshold (MEST) test. 2. Studies have now been undertaken to determine the effects of SB-204269 in a range of seizure models and tests of neurological deficits in rats. In addition, the compound has been evaluated in a series of in vitro mechanistic assays. 3. SB-204269 proved to be an orally-effective anticonvulsant agent, at doses (0.1-30 mg Kg-1) devoid of overt behavioural depressant properties, in models of both electrically (MEST and maximal electroshock (MEST)) and chemically (i.v. pentylenetetrazol (PTZ) infusion)-evoked tonic extension seizures. However, the compound did not inhibit PTZ-induced myoclonic seizures at doses up to 30 mg kg-1, p.o. 4. SB-204269 also selectively reduced focal electrographic seizure activity in an in vitro elevated K+ rat hippocampal slice model at concentrations (0.1-10 microM) that had no effect on normal synaptic activity and neuronal excitability. 5. In all of these seizure models, SB-204269 was equivalent or better than the clinically established antiepileptic drugs carbamazepine and lamotrigine, in terms of anticonvulsant potency and efficacy. 6. Unlike SB-204269, the corresponding trans 3S,4R enantiomer, SB-204268, did not produce marked anticonvulsant effects, an observation in accord with previous findings for other related pairs of trans enantiomers in the benzopyran series. 7. In the rat accelerating rotarod test, a sensitive paradigm for the detection of neurological deficits such as sedation and motor incoordination, SB-204269 was inactive even at doses as high as 200 mg kg-1, p.o. This was reflected in the excellent therapeutic index (minimum significantly effective dose in the rotarod test/ED50 in the MES test) for SB-204269 of > 31, as compared to equivalent values of only 7 and 13 for carbamazepine and lamotrigine, respectively. 8. At concentrations (> or = 10 microM) well above those required to produce anticonvulsant activity in vivo (i.e. 0.1 microM in brain), SB-204269 did not interact with many of the well known mechanistic targets for established antiepileptic drugs (e.g. Na+ channels or GABAergic neurotransmission). Subsequent studies have shown that the anticonvulsant properties of SB-204269 are likely to be mediated by a novel stereospecific binding site present in the CNS. 9. The overall efficacy profile in rodent seizure models, together with a minimal liability for inducing neurological impairment and an apparently unique mechanism of action, highlight the therapeutic potential of SB-204269 for the treatment of refractory partial and generalized tonic-clonic seizures.

Animals↗

PubMed1997

On the putative mechanistic basis for intraoperative propofol-induced penile erections.

Propofol is an hypnotic drug used in anesthesia which was noted to induce marked vasodilation in vivo and in vitro, and to elicit intraoperative penile erections. The goal of this study was to assess the putative mechanistic basis for this later observation by confirming its action in vivo in a rat model of penile erection, as well as by studying its effects in vitro on cultured human corporal smooth muscle cells and isolated corporal tissue strips. In vivo experiments were conducted on Fisher 344 rats anesthetized with sodium pentobarbital or propofol. Intracavernosal pressure was recorded during current stimulation of cavernous nerves. A significant increase in the intracavernous pressure response was recorded at all levels neurostimulation, ranging from 1-10 mA. In vitro experiments were conducted utilizing digital imaging microscopy to assess the effects of propofol (3-12 micrograms/mL) on ET-1-induced (50 nM) intracellular Ca2+ transients [Ca2+]i in Fura-2-loaded cultured human corporal smooth muscle cells (passage 3-4) as well as to evaluate the effects of propofol on phenylephrine (PE)-induced contractile responses on isolated corporal tissue strips. With respect to the former, resting cytosolic calcium levels were not altered during preincubation with propofol alone at clinically effective concentrations (12 micrograms/mL). However, propofol produced a concentration-dependent decrease in the peak amplitude of the transient ET-1-induced (50 nM) [Ca2+]i response (P < 0.001). Preincubation of the cells with calcium free/EGTA (1 mM) buffer produced a reduction in the peak amplitude of the ET-1-induced [Ca2+]i transient (55.5 +/- 6% (n = 10 cells, P < 0.01)) which was indistinguishable from that produced by 8 micrograms/mL of propofol (53.4 +/- 5.6% (n = 12 cells, P < 0.01)). However, propofol had no effect on the histamine-induced [Ca2+]i response. Lastly, preincubation of isolated human corporal tissue strips with propofol (100-200 microM; 30 min) caused a significant diminution in the peak amplitude of the PE-induced contractile response. Taken together, these data indicate that the mechanistic basis for intraoperative penile erections observed with propofol may be related, at least in part, to altered transmembrane calcium flux through voltage-dependent calcium channels, and thus, decreased corporal smooth muscle tone.

Adrenergic alpha-Agonists↗

PubMed1982

Mechanistic studies on C-19 demethylation in oestrogen biosynthesis.

Mechanistic aspects of the biosynthesis of oestrogen have been studied with a microsomal preparation from full-term human placenta. The overall transformation, termed the aromatization process, involves three steps using O(2) and NADPH, in which the C-19 methyl group of an androgen is oxidised to formic acid with concomitant production of the aromatic ring of oestrogen: [Formula: see text] To study the mechanism of this process in terms of the involvement of the oxygen atoms, a number of labelled precursors were synthesized. Notable amongst these were 19-hydroxy-4-androstene-3,17-dione (II) and 19-oxo-4-androstene-3,17-dione (IV) in which the C-19 was labelled with (2)H in addition to (18)O. In order to follow the fate of the labelled atoms at C-19 of (II) and (IV) during the aromatization, the formic acid released from C-19 was benzylated and analysed by mass spectrometry. Experimental procedures were devised to minimize the exchange of oxygen atoms in substrates and product with oxygens of the medium. In the conversion of the 19-[(18)O] compounds of types (II) and (IV) into 3-hydroxy-1,3,5-(10)-oestratriene-17-one (V, oestrone), it was found that the formic acid from C-19 retained the original substrate oxygen. When the equivalent (16)O substrates were aromatized under (18)O(2), the formic acid from both substrates contained one atom of (18)O. It is argued that in the conversion of the 19-hydroxy compound (II) into the 19-oxo compound (IV), the C-19 oxygen of the former remains intact and that one atom of oxygen from O(2) is incorporated into formic acid during the conversion of the 19-oxo compound (IV) into oestrogen. This conclusion was further substantiated by demonstrating that in the aromatization of 4-androstene-3,17-dione (I), both the oxygen atoms in the formic acid originated from molecular oxygen. 10beta-Hydroxy-4-oestrene-3,17-dione formate, a possible intermediate in the aromatization, was synthesized and shown not to be converted into oestrogen. In the light of the cumulative evidence available to date, stereochemical aspects of the conversion of the 19-hydroxy compound (II) into the 19-oxo compound (IV), and mechanistic features of the C-10-C-19 bond cleavage step during the conversion of the 19-oxo compound (IV) into oestrogen are discussed.

Androstenedione↗

PubMed2001

Splanchnic ischaemia-reperfusion injury: mechanistic insights provided by mutant mice.

Reperfusion of ischaemic tissues often leads to microvascular dysfunction that is manifested as impaired endothelium-dependent dilation of arterioles, enhanced fluid filtration and leucocyte plugging in capillaries, and the trafficking of leucocytes and plasma protein extravasation in postcapillary venules. Efforts to define the mechanisms that underlie these microvascular responses to ischaemia and reperfusion have largely relied on pharmacological agents and monoclonal antibodies. Gene-targeting technology has been applied to the production of transgenic and knockout mice that are rapidly gaining acceptance as tools for mechanistic studies of ischaemia-reperfusion (I/R) injury that obviate some of the concerns (e.g. specificity) raised about previously employed experimental strategies. This review summarizes some of our efforts to apply gene-targeted mice to the study of I/R injury in the splanchnic vascular bed. A role for endothelial cell adhesion molecules (CAMs) and reactive oxygen metabolites is supported by results from mutant mice. Low density lipoprotein receptor mice also reveal that the microvascular and inflammatory responses to I/R are greatly exaggerated during chronic hypercholesterolaemia. The wide variety of mutant mice that have been produced for inflammation-related research makes this experimental strategy particularly promising for mechanistic investigations of the tissue responses to I/R.

Animals↗

PubMedDate not supplied

Molecular and mechanistic validation of delayed healing rat wounds as a model for human chronic wounds.

The purpose of this study was to provide molecular and mechanistic evaluation of an ischemic wound model in rats to determine if it is a valid model for human chronic wounds. Compared to acute wounds, human chronic wounds contain markedly elevated levels of proinflammatory cytokines and matrix metalloproteinases, while matrix metalloproteinase inhibitors and growth factor activity are diminished. Accordingly, tissue from ischemic and normal rat wounds were analyzed for cytokine, proteases and growth factor levels. Dorsal full thickness punch wounds were created in rats using a reproducible template. The ischemic wound group (n = 10) had six uniformly placed wounds within a bipedicled dorsal flap. The control group (n = 10) had the same wounds created without elevation of a flap. On postwound days 3, 6 and 13 wounds were excised and analyzed. Protein levels for tumor necrosis factor-alpha were determined with a rat-specific enzyme-linked immunosorbent assay, while mRNA was determined by RNase protection assay. Matrix metalloproteinases and serine protease detection was done using gelatin and casein zymography, respectively. Significant delay in healing was achieved in the ischemic group: 50% healing for control wounds was at 7 days and 11 days for ischemic wounds (p < 0.001). No significant differences between wound groups were found for interleukin-1beta, and mRNA for tumor necrosis factor-alpha and interleukin-1beta. However, at day 13 ischemic wounds contained significantly more tumor necrosis factor-alpha than controls and normal skin (586 +/- 106 pg/biopsy vs. 79 +/- 7 pg/biopsy vs. 52 +/- 2 pg/biopsy; p < 0. 001). Zymography showed substantially greater quantities of matrix metalloproteinase-2, matrix metalloproteinase-9, and serine proteases in ischemic wounds. This model of delayed healing in rats shares many of the key biochemical, molecular and mechanistic characteristics found in human chronic wounds, namely elevated tumor necrosis factor-alpha and protease levels. As such, this model will likely prove to be useful in chronic wound research, particularly in developing novel therapeutics.

Animals↗

PubMedDate not supplied

A mechanistic study of evolvability using the mitogen-activated protein kinase cascade.

Evolvability is a function of the way genetic variation interacts with the mechanisms that produce the phenotype. We explore an explicitly mechanistic way of studying the evolvability of phenotypes that are produced by a relatively simple genetic mechanism, the mitogen-activated protein kinase (MAPK) cascade. We developed a quantitative model of MAPK activation that can be used to study the effects of genetic variation on the various components of this signaling cascade. We show how some standard tools of applied mathematics, such as steady-state formulations and nondimensionalization, can be used to elucidate the relative importance of variation in each gene of this mechanism. We also give insights into non-intuitive patterns of dependence and trade-off among the genes. The mechanism produces several different phenotypes (ultrasensitivity to stimulation, switch-like behavior, amount of MAPK-PP delivered, persistence of MAPK-PP activity), each of which is sensitive to different (but partially overlapping) combinations of genes. We show that the mechanism imposes clear limitations on the evolvability of each of the different phenotypes of the pathway, even in the presence of genetic variation in the components of the mechanism. This approach to the study of evolvability is generally applicable and complements the traditional approach through statistical genetics by providing a mechanistic understanding of the genetic interactions that produce the phenotype.

Biological Evolution↗

PubMed1999

The requirement for molecular chaperones during endoplasmic reticulum-associated protein degradation demonstrates that protein export and import are mechanistically distinct.

Polypeptide import into the yeast endoplasmic reticulum (ER) requires two hsp70s, Ssa1p in the cytosol and BiP (Kar2p) in the ER lumen. After import, aberrant polypeptides may be exported to the cytoplasm for degradation by the proteasome, and defects in the ER chaperone calnexin (Cne1p) compromise their degradation. Both import and export require BiP and the Sec61p translocation complex, suggesting that import and export may be mechanistically related. We now show that the cne1Delta and two kar2 mutant alleles exhibit a synthetic interaction and that the export and degradation of pro-alpha factor is defective in kar2 mutant microsomes. Pulse-chase analysis indicates that A1PiZ, another substrate for degradation, is stabilized in the kar2 strains at the restrictive temperature. Because two of the kar2 mutants examined are proficient for polypeptide import, the roles of BiP during ER protein export and import differ, indicating that these processes must be mechanistically distinct. To examine whether Ssa1p drives polypeptides from the ER and is also required for degradation, we assembled reactions using strains either containing a mutation in SSA1 or in which the level of Ssa1p could be regulated. We found that pro-alpha factor and A1PiZ were degraded normally, indicating further that import and export are distinct and that other cytosolic factors may pull polypeptides from the ER.

Adenosine Triphosphatases↗

PubMed2000

Anion-mediated iron release from transferrins. The kinetic and mechanistic model for N-lobe of ovotransferrin.

Iron release process of ovotransferrin N-lobe (N-oTf) to anion/chelators has been resolved using kinetic and mechanistic approach. The iron release kinetics of N-oTf were measured at the endosomal pH of 5.6 with three different anions such as nitrilotriacetate, pyrophosphate, and sulfate using stopped flow spectrofluorimetric method, all yielding clear biphasic progress curves. The two observed rate constants and the corresponding amplitudes obtained from the double exponential curve fit to the biphasic curves varied depending on the type and concentration of anions. Several possible models for the iron release kinetic mechanism were examined on the basis of a newly introduced quantitative equation. Results from the curve fitting analyses were consistent with a dual pathway mechanism that includes the competitive iron release from two different protein states, namely, X and Y, with the respective first order rate constants of K(1) and K(2) (X, domain closed holo N-oTf; Y, anion induced different conformer of holo N-oTf). The reversible interconversions of X to Y and Y to X are driven by the second order rate constant k(3) and the first order rate constant K(4), respectively. The obtained rate constants were greatly variable for the three anions depending on the synergistic or nonsynergistic nature. In the light of the anion-binding sites of N-oTf located crystallographically, the compatible mechanistic model that includes competitive anion binding to the iron coordination sites and to a specific anion site is suggested for the dual pathway iron release mechanism.

Anions↗

PubMed2003

Crystal structure and mechanistic implications of N2-(2-carboxyethyl)arginine synthase, the first enzyme in the clavulanic acid biosynthesis pathway.

The initial step in the biosynthesis of the clinically important beta-lactamase inhibitor clavulanic acid involves condensation of two primary metabolites, D-glyceraldehyde 3-phosphate and L-arginine, to give N2-(2-carboxyethyl)arginine, a beta-amino acid. This unusual N-C bond forming reaction is catalyzed by the thiamin diphosphate (ThP2)-dependent enzyme N2-(2-carboxyethyl)arginine synthase. Here we report the crystal structure of N2-(2-carboxyethyl)arginine synthase, complexed with ThP2 and Mg2+, to 2.35-A resolution. The structure was solved in two space groups, P2(1)2(1)2(1) and P2(1)2(1)2. In both, the enzyme is observed in a tetrameric form, composed of a dimer of two more tightly associated dimers, consistent with both mass spectrometric and gel filtration chromatography studies. Both ThP2 and Mg2+ cofactors are present at the active site, with ThP2 in a "V" conformation as in related enzymes. A sulfate anion is observed in the active site of the enzyme in a location proposed as a binding site for the phosphate group of the d-glyceraldehyde 3-phosphate substrate. The mechanistic implications of the active site arrangement are discussed, including the potential role of the aminopyrimidine ring of the ThP2. The structure will form a basis for future mechanistic and structural studies, as well as engineering aimed at production of alternative beta-amino acids.

Amino Acids↗