How do organic solvents affect peroxidase structure and function?

The effect of organic solvents on horseradish peroxidase structure and function has been studied. Some, but not complete, enzyme denaturation occurs even in low volumes of water-miscible organic solvents (e.g., greater than 30% v/v dioxane, greater than 50% v/v methanol, and greater than 20% v/v acetonitrile) as determined by the decreased difference between the fluorescence of peroxidase's sole tryptophan residue and free L-tryptophan in solution. Absorbance and electron paramagnetic resonance spectroscopies indicate exposure of peroxidase's active site to the organic solvent. This reduces the local polarity in the enzyme's active site and results in stronger hydrogen bonding of phenolic substrates to the enzyme. In extreme cases (e.g., 95% v/v dioxane, 90% v/v acetonitrile, and ethyl and butyl acetate containing 2 and 1% v/v aqueous buffer, respectively), the transition state of the enzymic reaction is sufficiently perturbed so as to alter the magnitude of the Hammett rho value. This is most likely the result of the increased strength of hydrogen bonding between electron-donating alkoxyphenols (negative sigma values) and an electrophilic group in the enzyme's active site, thereby reducing catalytic efficiencies for such substrates relative to alkyl- and chlorophenols. Perhaps the most important effect of the organic solvent, however, is the significant ground-state stabilization of phenolic substrates in organic media as opposed to aqueous buffer. This stabilization can account for nearly 4 orders of magnitude in reduction of catalytic efficiency and is manifested in increased Km's. This study indicates that enzymes can maintain much of their native active-site structure in organic media and that the effect of solvent on substrate thermodynamics must be considered.     

Why do bacteria use so many enzymes to scavenge hydrogen peroxide?

Hydrogen peroxide (H(2)O(2)) is continuously formed by the autoxidation of redox enzymes in aerobic cells, and it also enters from the environment, where it can be generated both by chemical processes and by the deliberate actions of competing organisms. Because H(2)O(2) is acutely toxic, bacteria elaborate scavenging enzymes to keep its intracellular concentration at nanomolar levels. Mutants that lack such enzymes grow poorly, suffer from high rates of mutagenesis, or even die. In order to understand how bacteria cope with oxidative stress, it is important to identify the key enzymes involved in H(2)O(2) degradation. Catalases and NADH peroxidase (Ahp) are primary scavengers in many bacteria, and their activities and physiological impacts have been unambiguously demonstrated through phenotypic analysis and through direct measurements of H(2)O(2) clearance in vivo. Yet a wide variety of additional enzymes have been proposed to serve similar roles: thiol peroxidase, bacterioferritin comigratory protein, glutathione peroxidase, cytochrome c peroxidase, and rubrerythrins. Each of these enzymes can degrade H(2)O(2) in vitro, but their contributions in vivo remain unclear. In this review we examine the genetic, genomic, regulatory, and biochemical evidence that each of these is a bonafide scavenger of H(2)O(2) in the cell. We also consider possible reasons that bacteria might require multiple enzymes to catalyze this process, including differences in substrate specificity, compartmentalization, cofactor requirements, kinetic optima, and enzyme stability. It is hoped that the resolution of these issues will lead to an understanding of stress resistance that is more accurate and perceptive.     

Biocatalytic synthesis of acrylates in organic solvents and supercritical fluids: III. Does carbon dioxide covalently modify enzymes?

We have previously demonstrated that the activity of the lipase (Candida cylindracea) catalyzed transesterification reaction between methylmethacrylate and 2-ethylhexanol in supercritical carbon dioxide is comparatively low. In this article, we have investigated the same reaction in supercritical carbon dioxide with a special emphasis on determining the extent of any interaction between the enzyme and carbon dioxide. Transesterification reaction rates in hexane and supercritical carbon dioxide are compared at different temperatures. In supercritical carbon dioxide, temperature was found to have no significant effect on reaction rate in the range of 40 degrees to 55 degrees C. Above 55 degrees C, however, the reaction rate increased significantly as a function of temperature. It appears that carbon dioxide forms reversible complexes with the free amine groups on the surface of the enzyme. Direct evidence of modification was obtained using mass spectroscopy to detect the extent of modification of a pure protein. The kinetics of the reaction have been studied in hexane, and they obey a ping-pong bi-bi mechanism with inhibition by 2-ethylhexanol. The effect of bubbling carbon dioxide and/or fluoroform on the reaction rate in hexane at different temperatures suggests that the enzyme undergoes shear inactivation in hexane. (c) 1995 John Wiley & Sons, Inc.     

Do organic solvents affect the catalytic properties of lipase? Intrinsic kinetic parameters of lipases in ester hydrolysis and formation in various organic solvents

When it is assumed that organic solvents do not interfere with the binding process nor with the catalytic mechanism, the contribution of substrate-solvent interactions to enzyme kinetics can be accounted for by just replacing substrate concentrations in the equations by thermodynamic activities. It appears from the transformation that only the affinity parameters (K(m), K(sp)) are affected by this. Thus, in theory, the values of these corrected, intrinsic parameters (K(m) (int), k(sp) (int)) and the maximal rate (V(1)) should be equal for all media. This was tested for hydrolysis, transesterification, and esterification reactions catalyzed by pig pancreas lipase and Pseudomonas cepacia lipase in various organic solvents. Correction was carried out via experimentally determined activity coefficients for the substrates in these solvents or, if not feasible, from values in data bases. However, although the kinetic performances of each enzyme in the solvents became much more similar after correction, differences still remained. Analysis of the enzyme suspensions revealed massive particles, which explains the low activity of enzymes in organic solvents. However, no correlation was found between estimates of the amount of catalytically available enzyme (present at the surface of suspended particles or immobilized on beads) and the maximal rates observed. Moreover, the solvents had similar effects on the intrinsic parameters of suspended and immobilized enzyme. The possible causes for the effects of the solvents on the catalytic performance of the enzymes, remaining after correction for solvent-substrate interactions and the amount of participating enzyme, are discussed with respect to the premises on which the correction method is based. (c) 1995 John Wiley & Sons, Inc.     

Why do stigmas move in a flexistylous plant?

Flexistyly is a recently documented stylar polymorphism involving both spatial and temporal segregation of sex roles within hermaphroditic flowers. Using the experimental manipulation of stigma movement in self-compatible Alpinia mutica, we tested the hypothesis that selection for reducing interference between male and female function drives the evolution and/or maintenance of stigma movement. In experimental arrays, anaflexistylous (protogynous) flowers served as pollen donors competing for mating opportunities on cataflexistylous (protandrous) flowers. The pollen donors were either manipulated so their stigmas could not move or were left intact, and their success was determined using allozymes to assess the paternity of recipient seeds. We found that manipulated flowers sired a significantly smaller proportion of seeds, showing that stigma movement in unmanipulated plants increased male fitness. This result was strongest under conditions in which pollen competition was expected to be highest, specifically when pollinators visited multiple donor plants before visiting recipient flowers.     

Why do Luehdorfia butterflies lay eggs in clusters?

Luehdorfia butterflies lay eggs in clusters. Clones of their host plants (Asiasarum and Heterotropa) are distributed pacthily among the understory of deciduous forests. Groups of Luehdorfia larvae often exhaust the clones and may wander over the forest floor seeking new clones. The highest mortality observed is during this wandering period. To elucidate why Luehdorfia butterflies lay eggs in clusters, a simulation experiment was made for hypothetical populations which lay eggs in clusters or singly. Field data on larval mortality, consumption, density of host clones and leaf weight for Luehdorfia japonica were incorporated into the model. The predictions of the simulation were: (1) When the egg density is low, the single egg type could leave many more pupae than the egg clustering type, but when the egg density is high, the former might leave smaller number of pupae than the latter; and (2) There are optimal sizes of egg clusters for different egg densities and the optimal size becomes larger as the egg density increased.     

Why do animals hybridize?

Animal hybridization is increasingly recognized as common and evolutionarily important, but the role of behavior in promoting hybridization events is not well understood. Understanding the behavioral causes of hybridization requires understanding the ecological, demographic, and phenotypic influences on mate choice in hybridizing taxa. Here, I review how these influences on mate choice can contribute to hybridization by (1) circumventing (female) choice, (2) bringing formerly isolated species into sympatry, (3) masking cues important for mate recognition, (4) altering the traits or preferences involved in mate recognition, or (5) altering the costs and benefits of mate choice. In particular, hybridization in response to either high direct costs of mate choice or high direct benefits of heterospecific mating may be widespread, a possibility that awaits further testing. Adopting a mate choice perspective to the study of hybridization challenges the assumption of hybrids as reproductive “mistakes” and allows for functional explanations regarding the occurrence and maintenance of hybridization. As evidence of the prevalence and evolutionary importance of animal hybridization accumulates, investigations into the role of behavior will be increasingly important for our understanding of diversification and for conservation applications.     

Why do plants silicify?

Despite seminal papers that stress the significance of silicon (Si) in plant biology and ecology, most studies focus on manipulations of Si supply and mitigation of stresses. The ecological significance of Si varies with different levels of biological organization, and remains hard to capture. We show that the costs of Si accumulation are greater than is currently acknowledged, and discuss potential links between Si and fitness components (growth, survival, reproduction), environment, and ecosystem functioning. We suggest that Si is more important in trait-based ecology than is currently recognized. Si potentially plays a significant role in many aspects of plant ecology, but knowledge gaps prevent us from understanding its possible contribution to the success of some clades and the expansion of specific biomes.     

L-Dopa ester synthesis catalyzed by α-chymotrypsin in organic solvents

Summary Esters of N-unprotected amino acids have been shown to be suitable substrates for -chymotrypsin-catalyzed reactions in organic solvents. Based on this observation, a wide range of L-Dopa esters were synthesized via enzymic transesterification, and up to 50% yields were obtained using various alcohols as acyl acceptors.     

Why do salmonid antipredator responses weaken in hatchery rearing?

The Arctic charr of Lake Saimaa are the most endangered fish population in Finland, and reintroduction programs have been unsuccessful. Low success of reintroduction programs has drawn attention to behavioural properties of hatcheryreared fish. Mortality due to predation often is a principal cause of failure. Antipredator behaviour may degenerate rapidly under hatchery conditions due to (i) reduced genetic variation in antipredator behaviour and/or (ii) selection that would favour bold and fast growing individuals and disfavour predator awareness supposedly associated with slow growth. To test the relative importance of these two factors we first analysed the amount of variation in innate antipredator responses between and within families of hatchery‐bred Arctic charr of the Lake Saimaa stock. We then tested whether fast growing individuals would show reduced responses to chemical cues from their natural predators compared to their slow growing counterparts. Based on the results we propose procedures for maintaining and improving antipredator skills of hatchery‐reared salmonids.     

Why do in utero stem cell transplants sometimes fail?

In utero stem cell transplantation promises a novel therapeutic approach to those genetic disorders that can be diagnosed in early pregnancy and that could lead to either severe disability or death. Available scientific evidence suggests that such procedures could achieve clinically relevant levels of engraftment with donor cells and that the resulting sustained chimerism is potentially long lived. However, the relatively few cases performed so far have not borne out initial hopes, and both the source and the type of cell preparation to be transplanted and the choice of disorders to target with this therapy remain controversial.     

So do we understand how enzymes work?

Between 1930 and 1975 biochemical and structural analysis of enzymes led to a clear set of ideas that might form a basis for detailed understanding of enzyme action. Further development required energetic and thermodynamic analysis of enzymes in an aqueous medium, beyond the computational power then available. Structural enzymology advanced in other directions, but the fundamental questions of enzyme action must soon be re-opened.     

Why do worms need cholesterol?

Cholesterol is a structural component of animal membranes that influences fluidity, permeability and formation of lipid microdomains. It is also a precursor to signalling molecules, including mammalian steroid hormones and insect ecdysones. The nematode Caenorhabditis elegans requires too little cholesterol for it to have a major role in membrane structure. Instead, its most probable signalling functions are to control molting and induce a specialized non-feeding larval stage, although no cholesterol-derived signalling molecule has yet been identified for these or any other functions.     

Why do microorganisms produce rhamnolipids?

We review the environmental role of rhamnolipids in terms of microbial life and activity. A large number of previous research supports the idea that these glycolipids mediate the uptake of hydrophobic substrates by bacterial cells. This feature might be of highest priority for bioremediation of spilled hydrocarbons. However, current evidence confirms that rhamnolipids primarily play a role in surface-associated modes of bacterial motility and are involved in biofilm development. This might be an explanation why no direct pattern of hydrocarbon degradation was often observed after rhamnolipids supplementation. This review gives insight into the current state of knowledge on how rhamnolipids operate in the microbial world.