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Mechanistic models for wind dispersal.

The growing need for ecological forecasts of, for example, species migration, has increased interest in developing mechanistic models for wind dispersal of seeds, pollen and spores. Analytical models are only able to predict mean dispersal distances, whereas sophisticated trajectory simulation models are able to incorporate rare wind conditions causing long-distance dispersal and are therefore preferable. Despite the rapid development of mechanistic dispersal models, only a few studies have focused on comparing the performance of the models. To assess the level of model complexity needed, attention should be paid to model comparisons and the sensitivity of the predictions to model complexity. In addition to studying the movement of airborne particles, future modelling work should also focus on the processes of particle release and deposition.

Models, Biological↗

Mechanistic implications of the equality of compensation temperatures in chromatography.

A common interpretation of the observation that two processes exhibit similar compensation temperatures in an enthalpy-entropy plot is that the two processes occur via the same "mechanism". We show that this interpretation is not rigorously allowed. In fact, the only thing that can be concluded from the observation of identical compensation temperatures is that the relative contributions of enthalpy and entropy to the overall free energy are the same in the two processes. Since it is possible that two processes occur via different mechanisms that, by chance, result in the same relative blends of enthalpy and entropy, the observation of identical compensation temperatures cannot be used as evidence for mechanistic identity. If two processes exhibit different compensation temperatures, however, it can logically be concluded that the two processes are mechanistically distinct.

Chromatography↗

Mechanistic studies on protein tyrosine phosphatases.

The human genome encodes approximately 100 phosphatases that belong to the protein tyrosine phosphatase (PTP) superfamily. The hallmark for this superfamily is the active site sequence C(X)5R, also known as the PTP signature motif. The PTPs are key regulatory components in signal transduction pathways and the importance of PTPs in the control of cellular signaling is well established. Based on structure and substrate specificity, the PTP superfamily is divided into four distinct subfamilies: (1) pTyr-specific PTPs, (2) dual specificity phosphatases, (3) Cdc25 phosphatases, and (4) LMW PTPs. The PTPs have similar core structures made of a central parallel beta-sheet with flanking a-helices containing a beta-loop-alpha-loop that encompasses the PTP signature motif. Site-directed mutagenesis of conserved amino acids in the Yersinia PTP and several other phosphatases in the PTP superfamily combined with detailed kinetic and mechanistic analyses have revealed a common chemical mechanism for phosphate hydrolysis despite the differences in substrate specificity. This article reviews our current knowledge of the common features important for PTP catalysis, the nature of the enzymatic transition state, and the roles of essential residues in transition stabilization. Future mechanistic studies of PTPs will focus on the use of physiological substrates to determine the molecular basis of substrate recognition and regulation, which is essential for understanding the specific functional role of PTPs in cellular signaling.

Amino Acid Sequence↗

The use of mechanistic data in the risk assessments of ten chemicals: an introduction to the chemical-specific reviews.

The International Expert Panel on Carcinogen Risk Assessment of the American Health Foundation has planned, directed and reviewed in-depth analyses of mechanistic data for 10 rodent carcinogens. The purpose of this review was to illustrate the use of mechanistic data in carcinogen risk assessment. The mechanisms by which a chemical produces cancer in rodents provided important information for determining whether or not humans would be at risk at low exposure levels. For epigenetic (non-DNA-reactive) carcinogens, current exposure levels for these chemicals below a threshold do not pose a risk. For DNA-reactive agents at sufficient exposures, humans would be expected to be at risk. However, protective mechanisms may lower the expected risk, especially at low-level exposures.

Animals↗

Respiratory cancer risks associated with low-level nickel exposure: an integrated assessment based on animal, epidemiological, and mechanistic data.

Increased lung and nasal cancer risks have been reported in several cohorts of nickel refinery workers, but in more than 90% of the nickel-exposed workers that have been studied there is little, if any evidence of excess risk. This investigation utilizes human exposure measurements, animal data from cancer bioassays of three nickel compounds, and a mechanistic theory of nickel carcinogenesis to reconcile the disparities in lung cancer risk among nickel-exposed workers. Animal data and mechanistic theory suggest that the apparent absence of risk in workers with low nickel exposures is due to threshold-like responses in lung tumor incidence (oxidic nickel), tumor promotion (soluble nickel), and genetic damage (sulfidic nickel). When animal-based lung cancer dose-response functions for these compounds are extrapolated to humans, taking into account interspecies differences in deposition and clearance, differences in particle size distributions, and human work activity patterns, the predicted risks at occupational exposures are remarkably similar to those observed in nickel-exposed workers. This provides support for using the animal-based dose-response functions to estimate occupational exposure limits, which are found to be comparable to those in current use.

Animals↗

Mechanistic equations for membrane substance transport and their identity with Kedem-Katchalsky equations.

Since the physical interpretation of practical Kedem-Katchalsky (KK) equations is not clear, we consider an alternative, mechanistic approach to membrane transport generated by osmotic and hydraulic pressure. We study a porous membrane with randomly distributed pore sizes (radii). We postulate that reflection coefficient (sigma(p)) of a single pore may equal 1 or 0. From this postulate we derive new (mechanistic) transport equations. Their advantage is in clear physical interpretation and since we show they are equivalent to the KK equations, the interpretation of the latter became clearer as well. Hence the equations allow clearer and more detailed interpretation of results concerning membrane substances transport.

Animals↗

Reactive oxygen species and biological aging: a mechanistic approach.

The experimental evidence for an age-dependent increased generation of reactive oxygen species and a progressive accumulation of oxidized biomolecules is growing. However, despite such facts there is no detailed mechanistic information on how the higher availability of reactive oxygen species translates into the accumulation of oxidized biomolecules. For example, open questions are which reactive oxygen species are responsible for what types of oxidation products in vivo, under what specific reaction conditions can we expect which reaction products, and why specifically are modified biomolecules eliminated whereas others accumulate? Mitochondria appear to serve as the major source for reactive oxygen species in aging tissue. Genetic experiments have demonstrated an effect of Cu,ZnSOD on life span and in the prevention of age-related oxidative damage, suggesting that extramitochondrial superoxide promotes biological aging. However, as superoxide does not easily cross membranes, potential chemical pathways that convert mitochondrial reactive oxygen species into superoxide outside the mitochondria are displayed. The chemical reactivity of individual reactive oxygen species with the amino acid side chain of methionine is surveyed to obtain mechanistic details on the oxidation pathways potentially leading to the age-dependent methionine oxidation of the protein calmodulin in vivo. It will evolve that the in vivo accumulation of oxidized calmodulin cannot be the result of the reaction of an individual reactive oxygen species with calmodulin in homogenous solution alone. Complexation of calmodulin to calmodulin-binding proteins and protein turnover are additional parameters likely contributing to the accumulation of specifically modified calmodulin.

Aging↗

Scope, limitations and mechanistic aspects of the photo-induced cross-linking of proteins by water-soluble metal complexes.

BACKGROUND: Chemical cross-linking is a valuable tool with which to study protein-protein interactions. Recently, a new kind of cross-linking reaction was developed in which the photolysis of associated proteins with visible light in the presence of ammonium persulfate and tris(2,2'-bipyridyl)ruthenium(II) dication or palladium(II) porphyrins results in rapid and efficient covalent coupling (Fancy, D.A. & Kodadek, T. (1999). Proc. Natl. Acad. Sci. USA 96, 6020-6024 and Kim, K., Fancy, D.A. & Kodadek, T. (1999). J. Am. Chem. Soc. 121, 11896-11897). Here, mechanistic and practical aspects of the reaction of importance for its application to biochemical problems are examined. RESULTS: It is shown that the photo-initiated cross-linking chemistry can be optimized for the analysis of protein-protein interactions in crude cell extracts. A number of commonly used epitope or affinity tags survive the reaction in functional form, allowing the simple visualization of the cross-linked products, or their isolation. It is shown that very little light-independent oxidation of protein residues occurs and that significant perturbation of complexes of interest prior to the brief photolysis period does not occur. Finally, evidence is presented that is consistent with a mechanistic model in which ammonium persulfate functions simply as an electron acceptor, facilitating the generation of the key high valent metal complex from the photoexcited species by electron transfer. In the absence of an electron acceptor, a much lower efficiency reaction is observed that appears to involve products resulting from reaction of the excited state metal complex with molecular oxygen. CONCLUSIONS: These results provide useful practical information for chemists and biochemists who may wish to employ this new cross-linking chemistry for the analysis of protein complexes. They also shed new light on the mechanism of this interesting reaction.

Bacterial Proteins↗

Surgery for malignant gliomas: mechanistic reasoning and slippery statistics.

Current surgical treatment of malignant gliomas largely depends on mechanistic reasoning and data collected in non-randomised studies. Technological advance has enabled more accurate resection of tumours and preservation of eloquent brain areas but ethical considerations have restricted randomised trials on the efficacy of surgery to one small trial that found a 3 month survival advantage for patients over age 65 years who received surgery and interim analysis of a larger trial. There is an argument for surgery as a palliative measure in patients with symptoms caused by mechanisms that are surgically remediable. Whether there is any survival advantage from surgery in patients other than those with immediately life-threatening, surgically remediable complications, such as raised intracranial pressure, is unclear. The available data show that if such an advantage does exist, it is modest at best. Adjuvant treatments given surgically are being studied. Chemotherapy wafers are the most prominent of the adjuvant treatments but the evidence available is insufficient to recommend their use in routine practice. In this review we examine the prevailing mechanistic model and observational data; we assess how these are applied and the priorities they indicate for future research.

Biopsy↗

Metabolism of the lactating cow. III. Properties of mechanistic models suitable for evaluation of energetic relationships and factors involved in the partition of nutrients.

Two models of lactating dairy cows were constructed by combining mechanistic models of digestion and metabolism reported previously and by adding elements to allow simulation of changes in tissue metabolic capacities over time. One model (day version) had an integration interval of 0.005 d and was suitable for simulation of within-day dynamics of nutrient supply and partition. The other (lactation version) had an integration interval of 1 d and was suitable for simulation of full lactation cycles. A number of simulation analyses were conducted to characterize and evaluate the models, to examine quantitative and dynamic properties of mechanisms which influence partition of nutrients, to identify aspects requiring further study, and to illustrate the potential usefulness of mechanistic, as compared to empirical, models in analyses of energy balance in lactating dairy cows.

Animal Nutritional Physiological Phenomena↗

Toward a mechanistic psychology of dialogue.

Traditional mechanistic accounts of language processing derive almost entirely from the study of monologue. Yet, the most natural and basic form of language use is dialogue. As a result, these accounts may only offer limited theories of the mechanisms that underlie language processing in general. We propose a mechanistic account of dialogue, the interactive alignment account, and use it to derive a number of predictions about basic language processes. The account assumes that, in dialogue, the linguistic representations employed by the interlocutors become aligned at many levels, as a result of a largely automatic process. This process greatly simplifies production and comprehension in dialogue. After considering the evidence for the interactive alignment model, we concentrate on three aspects of processing that follow from it. It makes use of a simple interactive inference mechanism, enables the development of local dialogue routines that greatly simplify language processing, and explains the origins of self-monitoring in production. We consider the need for a grammatical framework that is designed to deal with language in dialogue rather than monologue, and discuss a range of implications of the account.

Humans↗

Synthetic and mechanistic studies of the cycloisomerization and cyclization/hydrosilylation of functionalized dienes catalyzed by cationic palladium(II) complexes.

This Account describes the development and mechanistic study of the cycloisomerization and cyclization/hydrosilylation of functionalized dienes catalyzed by cationic palladium(II) complexes. These transformations are characterized by good functional group compatibility, high regio- and stereoselectivity, and low air- and moisture-sensitivity. Mechanistic studies of palladium phenanthroline-catalyzed diene cycloisomerization and cyclization/hydrosilylation established mechanisms involving C-C bond formation via intramolecular carbometalation of an alkyl olefin chelate complex, which was directly observed in the context of cyclization/hydrosilylation.

Catalysis↗

Mechanistic investigation of smart polymer-protein conjugates.

Many affinity separation and diagnostic applications rely upon both capture and release steps. There is thus a need for methods to enhance the reversibility of biomolecular interactions. We have previously demonstrated that stimuli-responsive polymers can be used to gate biomolecular reactions when conjugated near the active site of proteins. Here we have used a new smart polymer, N,N-dimethyl acrylamide-co-4-phenylazophenylacrylate that has allowed a mechanistic investigation of the smart polymer switches. This polymer was conjugated via a vinyl sulfone terminus to cysteine residues of genetically engineered streptavidin mutant E116C, where the polymer is conjugated close to the biotin-binding site, and streptavidin mutant S139C, where the conjugation site is distant. The biotin binding switching activity was strongly dependent on conjugation position, as the E116C conjugate displayed a large thermal response while the S139C conjugate displayed only small effects. Kinetic measurements of biotin release demonstrated that the off-rate of biotin was unperturbed and that the thermally triggered release of biotin with the E116C conjugate was due to the blocking the reassociation of biotin. The addition of free polymer to purified E116C conjugates was also shown to increase the blocking and release properties of the switch. This effect was site dependent, suggesting that the conjugated polymers were directing a physical aggregation near the binding site that effectively enhanced the switching activity. These investigations provide mechanistic insight that can be utilized to design better molecular switches for a variety of stimuli-responsive polymer-protein conjugates.

Acrylates↗

Isoabzymes: structurally and mechanistically similar catalytic antibodies from the same immunization.

Mechanistic and structural comparisons of five catalytic monoclonal antibodies generated from the same hybridoma fusion indicated that all five hydrolyze phenyl acetate by subtle variations of the same mechanism. All of the antibodies showed a pre-steady-state multi-turnover burst in which kcat and Km declined but kcat/Km did not change. The burst of one of the antibodies, 20G9, has previously been found to result from inhibition by the product, phenol. Although all of the antibodies showed the burst, their individual values for kcat, Km, and hapten Ki differed substantially. Three of the antibodies that were investigated for the effect of pH on kcat showed an acid limb pK of 9.5-9.6. Substrate inhibition was seen in four of the five antibodies. Variable region nucleotide sequencing of the heavy and light chains confirmed that all five antibodies were structurally similar and also revealed several potentially critical tyrosines. Despite their structural similarities, analysis of their sequences suggested that the antibodies are products of distinct, independent rearrangements of immunoglobulin gene segments that took place in different progenitor B cells. A plot of Ki for hapten inhibition vs Km/kcat for substrate hydrolysis for the mechanistically related antibodies ("isoabzymes") gave a linear relationship suggesting a catalytic role for transition-state complementarity. Taken together with previous work [Martin et al. (1991) Biochemistry 30, 9757-9761], the data conform to a mechanism in which the antibodies exploit both transition-state complementarity and an acyl-tyrosyl intermediate during phenyl acetate hydrolysis.

Amino Acid Sequence↗

Mechanistic diversity in a metalloenzyme superfamily.

It is now appreciated that the relationships of proteins, particularly enzymes, within a protein superfamily can be understood not only in terms of their sequence similarities and three-dimensional structures but also by chemical threads that relate their functional attributes. The mechanistic ties among superfamily members can often be traced to a common transition state for the rate-limiting step of the reactions being catalyzed. This paper presents an analysis of a metalloenzyme superfamily, the members of which catalyze a very diverse set of reactions with unrelated transition states but a more general common mechanistic imperative. The vicinal oxygen chelate (VOC) superfamily is composed of structurally related proteins with paired beta alpha beta beta beta motifs that provide a metal coordination environment with two or three open or readily accessible coordination sites to promote direct electrophilic participation of the metal ion in catalysis. The known types of reactions that are catalyzed include isomerizations (glyoxalase I), epimerizations (methylmalonyl-CoA epimerase), oxidative cleavage of C-C bonds (extradiol dioxygenase), and nucleophilic substitutions (fosfomycin resistance proteins). The remarkable access to mechanism space that is provided by the VOC superfamily appears to derive from a simple, pseudosymmetric structural fold that maximizes the catalytic versatility of the metal center.

Acetaldehyde↗

Mechanistic studies of peptidyl prolyl cis-trans isomerase: evidence for catalysis by distortion.

Cyclophilin, the cytosolic binding protein for the immunosuppressive drug cyclosporin A, has recently been shown to be identical with peptidyl prolyl cis-trans isomerase [Fischer, G., Wittmann-Liebold, B., Lang, K., Kiefhaber, T., & Schmid, F.X. (1989) Nature 337, 476; Takahashi, N., Hayano, T., & Suzuki, M. (1989) Nature 337, 473]. To provide a mechanistic framework for studies of the interaction of cyclophilin with cyclosporin, we investigated the mechanism of the PPI-catalyzed cis to trans isomerization of Suc-Ala-Xaa-cis-Pro-Phe-pNA (Xaa = Ala, Gly). Our mechanistic studies of peptidyl prolyl cis-trans isomerase include the determination of steady-state kinetic parameters, pH and temperature dependencies, and solvent and secondary deuterium isotope effects. The results of these experiments support a mechanism involving catalysis by distortion in which the enzyme uses free energy released from favorable, noncovalent interactions with the substrate to stabilize a transition state that is characterized by partial rotation about the C-N amide bond.

Amino Acid Isomerases↗

Kinetic and mechanistic studies of penicillin-binding protein 2x from Streptococcus pneumoniae.

High molecular weight penicillin-binding proteins (PBPs) are bifunctional enzymes that build bacterial cell walls from the glycopeptide lipid II [GlcNAc-MurNAc(L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Ala)-pyrophosphate-undecaprenol] by a process involving disaccharide polymerization and peptide cross-linking. The latter reaction involves acyl-transfer chemistry in which the penultimate (D)Ala first acylates the active-site serine, with release of the terminal (D)Ala, and is then transferred to the epsilon-amine of a Lys on a neighboring pentapeptide chain. These enzymes also catalyze hydrolysis of specific thioester substrates and acylation by beta-lactam antibiotics. In this paper, we explore these latter two reactions and report mechanistic experiments on the reaction of Streptococcus pneumoniae PBP 2x with N-benzoyl-(D)Ala-thioacetic acid [Bz-(D)Ala-(S)Gly] and penicillin G. For these experiments, we used PBP 2x, a soluble form of PBP 2x in which the transmembrane domain was deleted. The following results are significant: (1) pH dependencies for acylation of PBP 2x by penicillin G and Bz-(D)Ala-(S)Gly are identical, suggesting that the same ionizable residues are involved in both reactions and that these residues play the same catalytic role in the two processes. On the basis of these results, we propose a mechanistic model that is also consistent with recently published structural data [Gordon, E., et al. (2000) J. Mol. Biol. 299, 477-485]. (2) Pre-steady-state experiments for the PBP 2x-catalyzed hydrolysis of Bz-(D)Ala-(S)Gly at pH 6.5 indicate that k(c) is principally rate-limited by acylation with some contribution from deacylation. The contribution of these steps to rate limitation is pH-dependent, with acylation entirely rate-limiting at pH values less than 5.5 and deacylation principally rate-limiting above pH 8.5. (3) Results of solvent isotope effect and proton inventory experiments for acylation suggest a complex process that is at least partially rate-limited by chemistry with some involvement of changes in solvation and/or enzyme conformation. (4) Analysis of activation parameters suggests that during the acylation of PBP 2x by penicillin G the inherent chemical stability of penicillin's amide bond, as manifested in the enthalpy of activation, is offset by a favorable entropy term that reflects penicillin's rotationally constrained bicyclic system, which presumably allows a less energetically demanding entry into the transition state for acylation.

Acylation↗

Mechanistic significance of the preparatory migration of hydrogen atoms around the FeMo-co active site of nitrogenase.

The migration of H atoms over S and Fe atoms in the reaction domain of FeMo-co, the active site of nitrogenase, is described and used to explain mechanistic data on the catalyzed reductions of N(2) and C(2)H(2). After electron transfer to FeMo-co, H atoms are generated by fast proton supply to S3B (atom labels from structure 1M1N) and migrate vectorially via several pathways from S3B to locations on the FeMo-co face, specifically Fe6, S2B, Fe2, and S2A (calculated reaction profiles are reported). The E(n)H(n) reduction levels (n = 1-4) in the Thorneley-Lowe kinetic-mechanistic schemes are each potential sequences of substructures with different distributions of H atoms. The positions of H atoms influence the binding of substrates N(2) and C(2)H(2), and the bound substrate subsequently blocks further migration of H atoms past the binding site. This model provides a consistent structural interpretation of (a) the two-site reactivity of C(2)H(2) and the differentiation of the high- and low-affinity sites as due to different preparatory H migration; (b) the differing mutual inhibitions of N(2) and C(2)H(2) in wild-type protein; (c) the modified reactivity of the Azotobacter vinelandii alpha-(Gly)69(Ser) mutant with N(2) and C(2)H(2); and (d) the basis for the stereoselectivity of hydrogenation of C(2)D(2) and its loss in some mutant proteins. Some structures for initially bound N(2) and C(2)H(2), and their hydrogenated intermediates, are presented. The key new concept is that binding sites and binding states for substrates and intermediates are characterized not only by their locations on the FeMo-co face but also by the structural and temporal status of the distribution of H atoms over the FeMo-co reaction domain.

Acetylene↗