Categorization of enzyme deactivations using a series-type mechanism

A series-type enzyme deactivation model involving an active enzyme precursor and a final enzyme state with possible non-zero activity is proposed to categorize enzyme deactivation curves. The enzyme activity is a weighted function of the active enzyme states. The deactivation curves may be broadly classified into two major categories wherein the activity is either always less than or it may be more than the initial activity for some time period. Data taken from the literature may be classified into 14 cases. Complex enzyme deactivation curves exhibiting enzyme stabilization and a flex are some of the features that are classified.     

Influence of pH on enzyme stabilization: an analysis using a series-type mechanism

A series-type model is utilized to show the influence of pH on enzyme inactivation kinetics and stability. Examples of enzyme inactivations involving both single-step and series-type mechanisms are presented. Empirical relations for the inactivation rate constant for the first step and the residual activity as a function of pH are presented. This provides physical insights into the enzyme inactivation processes. The analysis forms the beginning of a framework within which one could quantitatively manipulate the inactivation rate constants and the residual activity for enzymes in desired directions as a function of pH.     

Series-type enzyme deactivations: Influence of intermediate activity on deactivation kinetics

A series-type enzyme deactivation model involving an active enzyme precursor is proposed wherein the enzyme activity is a weighted function of the active enzyme states. The active enzyme precursor may be less active, as active or more active than the initial enzyme form. The proposed model is shown to fit the soluble and immobilized enzyme deactivation data presented reasonably well. Some enzymes exhibit a ‘compensation-like’ effect. In other enzymes, if the deactivation rate coefficient for the second step, k₂, is zero, then the activity may stabilize to a value that depends upon the relative activities of the two active enzyme states.     

A mathematical analysis of enzyme stabilization by a series-type mechanism: Influence of chemical modifiers

A series-type enzyme deactivation model is utilized to theoretically analyze and to quantify the effect of chemical modifier concentration on the eventual level of enzyme activity stabilization, alpha(2). An increase in the concentration of phosphate ion and NADP increases alpha(2) for the enzymes studied. One example of each enzyme deactivation is given wherein the introduction of chemical modifiers changes the deactivation mechanism from a single-step to a series-type mechanism, and from a series-type to a single-step mechanism. Simple empirical equations are proposed to quantify the effect of chemical modifier concentration on alpha(2).     

Mechanistic and kinetic analysis of the DcpS scavenger decapping enzyme

Decapping is an important process in the control of eukaryotic mRNA degradation. The scavenger decapping enzyme DcpS functions to clear the cell of cap structure following decay of the RNA body by catalyzing the hydrolysis of m(7)GpppN to m(7)Gp and ppN. Structural analysis has revealed that DcpS is a dimeric protein with a domain-swapped amino terminus. The protein dimer contains two cap binding/hydrolysis sites and displays a symmetric structure with both binding sites in the open conformation in the ligand-free state and an asymmetric conformation with one site open and one site closed in the ligand-bound state. The structural data are suggestive of a dynamic decapping mechanism where each monomer could alternate between an open and closed state. Using transient state kinetic studies, we show that both the rate-limiting step and rate of decapping are regulated by cap substrate. A regulatory mechanism is established by the intrinsic domain-swapped structure of the DcpS dimer such that the decapping reaction is very efficient at low cap substrate concentrations yet regulated with excess cap substrate. These data provide biochemical evidence to verify experimentally a dynamic and mutually exclusive cap hydrolysis activity of the two cap binding sites of DcpS and provide key insights into its regulation.     

Mechanistic investigation of a starch-branching enzyme using hydrodynamic volume SEC analysis

Two linear alpha-(1,4)-D-glucans substrates, of degrees of polymerization DP approximately 150 and 6000, were exposed to maize starch-branching enzyme IIa (mSBEIIa) in vitro. The resulting branched alpha-glucans and their constituent chains (obtained by debranching) were analyzed by nuclear magnetic resonance (NMR) and size-exclusion chromatography (SEC). SEC data for the debranched species are presented as chain-length distributions, while those for branched species are presented as hydrodynamic volume distributions (HVDs), which is the most meaningful way to present such data (because SEC separates by size, not molar mass, and a sample of branched polymers with the same size can have a range of molar masses). A rigorous interpretation of the HVDs of the substrate and its branched product show that at least part of the branching is an interchain transfer mechanism in both the short- and long-chain substrate cases. A bimodal HVD of the in vitro branched alpha-glucan derived from the short-chain substrate was observed, and it is postulated that the divergence of the two populations is due to very small chains being unable to undergo branching. In the case of the in vitro branching of the long-chain substrate, the formation of maltohexaose during the reaction and the presence of a monomodal HVD were observed, suggesting a distinct mode of action of mSBEIIa on this substrate. Quantification of the branching level by NMR showed the branched glucans from both substrates had substantial amounts of branching (2.1-4.5%), ascribed to the intrinsic nature of the action of mSBEIIa on the two substrates. It is postulated that differences in the degrees of substrate association affect the pattern of branching catalyzed by the enzyme, and a putative active site structure is proposed based on the appearance of maltohexaose. The molar mass distribution of the constituent chains of the in vitro branched alpha-glucans obtained by isoamylase treatment reveals the transfer of chains of specific size and supports the supposition given in the literature that mSBEIIa is responsible for short-chain branching in amylopectin. It is suggested that hydrodynamic volume SEC analysis should be used as a tool for the mechanistic investigation of SBEs, allowing SEC data of in vitro branched alpha-glucans to be both comparable and quantitative.     

Kinetic analysis of the RNAi enzyme complex

The siRNA-directed ribonucleoprotein complex, RISC, catalyzes target RNA cleavage in the RNA interference pathway. Here, we show that siRNA-programmed RISC is a classical Michaelis-Menten enzyme in the presence of ATP. In the absence of ATP, the rate of multiple rounds of catalysis is limited by release of the cleaved products from the enzyme. Kinetic analysis suggests that different regions of the siRNA play distinct roles in the cycle of target recognition, cleavage, and product release. Bases near the siRNA 5' end disproportionately contribute to target RNA-binding energy, whereas base pairs formed by the central and 3' regions of the siRNA provide a helical geometry required for catalysis. Finally, the position of the scissile phosphate on the target RNA seems to be determined during RISC assembly, before the siRNA encounters its RNA target.     

The influence of insulin on various enzyme activities in human and rat hepatoma cells.

1. Incubation of human and rat hepatoma cells with insulin (1 mU/10(6) cells) decreases their content of adenosine 3':5'-monophosphate by more than half after 1 h and by about a quarter after 4 h. 2. The activities of the ATP-metabolising enzymes, adenylate kinase and Mg2+-adenosine triphosphatase are significantly increased by insulin within 1 h and after 4 h. Activity of succinate dehydrogenase and lactic dehydrogenase showed no change at either time interval. 3. Insulin markedly stimulated glucose 6-phosphate dehydrogenase activity within 1 h but by 4 h the increase was less apparent. Glutamate dehydrogenase activity by contrast was not increased by 1 h but was elevated at 4 h.     

Anaerobic acidogenesis of a complex wastewater: I. The influence of operational parameters on reactor performance

The influence of operational, parameters, such as hydraulic retention time, organic loading rate, influent substrate concentration, pH, and temperature, on the performance of the first phase of anaerobic digestion has been investigated. A complex substrate based on beef extract was used, and six series of experimental runs were conducted, each one showing the effect of one operational variable. The predominant fermentation products were always acetic and propionic acid, independent of the values of the operational parameters. For initial COD concentrations and hydraulic retention times above the critical values identified as 3 g/L and 6 h, respectively, the degree of acidification achieved was between 30 and 60%. The degree of acidification was found to increase with the hydraulic retention time and decrease with the influent substrate concentration and organic loading rate, while the opposite held true for the rate of product formation. Furthermore, it has been demonstrated that acidification is primarily determined by the hydraulic retention time and the rate of product formation by the influent substrate concentration. The concentration of the acetic acid produced was found to depend on the operational parameters. However, the concentration of propionic acid produced depended only on the substrate availability with a consistent proportion of 8% initial COD converted to it. The optimum pH and temperature were 7 and 40 degrees C, respectively. The percentage of acetic acid as a proportion of the total volatile fatty acids produced was found to increase with increasing pH and temperature, while the percentage of propionic acid seemed to decrease accordingly. Finally the effect of the temperature on the rate of acidification followed an Arrhenius type equation with an activation energy equal to 4739 cal/mol.     

Mechanistic studies of melanogenesis: the influence of N-substitution on dopamine quinone cyclization

The influence of side-chain structure on the mode of reaction of ortho-quinone amines has been investigated with a view, ultimately, to developing potential methods of therapeutic intervention by manipulating the early stages of melanogenesis. Four N-substituted dopamine derivatives have been prepared and quinone formation studied using pulse radiolysis and tyrosinase-oximetry. Ortho-quinones with an amide or urea side chain were relatively stable, although evidence for slow formation of isomeric para-quinomethanes was observed. A thiourea derivative cyclized fairly rapidly (k = 1.7/s) to a product containing a seven-membered ring, whereas a related amidine gave more rapidly (k approximately 2.5 x 10(2)/s) a stable spirocyclic product. The results suggest that cyclization of amides, ureas and carbamates (NHCO-X; X = R, NHR or OR) does not occur and is not, therefore, a viable approach to the formation of tyrosinase-activated antimelanoma prodrugs. It is also concluded that for N-acetyldopamine spontaneous ortho-quinone to para-quinomethane isomerization is slow.     

Structural analysis of urate oxidase in complex with its natural substrate inhibited by cyanide: Mechanistic implications

Background  

Urate oxidase (EC 1.7.3.3 or UOX) catalyzes the conversion of uric acid and gaseous molecular oxygen to 5-hydroxyisourate and hydrogen peroxide, in the absence of cofactor or particular metal cation. The functional enzyme is a homo-tetramer with four active sites located at dimeric interfaces.     

Mechanistic studies of a protonolytic organomercurial cleaving enzyme: bacterial organomercurial lyase

Mechanistic studies of the protonolytic carbon-mercury bond cleavage by organomercurial lyase from Escherichia coli (R831) suggest that the reaction proceeds via an SE2 pathway. Studies with stereochemically defined substrates cis-2-butenyl-2-mercuric chloride (1) and endo-norbornyl-2-mercuric bromide (2) reveal that a high degree of configurational retention occurs during the bond cleavage, while studies with exo-3-acetoxynortricyclyl-5-mercuric bromide (3) and cis-exo-2-acetoxy-bicyclo[2.2.1]hept-5-enyl-3-mercuric bromide (4) show that the protonolysis proceeds without accompanying skeletal rearrangement. Kinetic data for the enzymatic reactions of cis-2-butenyl-2-mercuric chloride (1) and trans-1-propenyl-1-mercuric chloride (6) indicate that these substrates show enhanced reaction rates of ca. 10-200-fold over alkylvinylmercurials and unsubstituted vinylmercurials, suggesting that the olefinic methyl substituent may stabilize an intermediate bearing some positive charge. Enzymatic reaction of 2-butenyl-1-mercuric bromide (5) yields a 72/23/5 mixture of 1-butene/trans-2-butene/cis-2-butene, indicative of intervening SE2' cleavage. The observation of significant solvent deuterium isotope effects at pH 7.4 of Vmax (H2O)/Vmax(D2O) = 2.1 for cis-2-butenyl-2-mercuric chloride (1) turnover and Vmax(H2O)/Vmax(D2O) = 4.9 for ethylmercuric chloride turnover provides additional support for a kinetically important proton delivery. Finally, the stoichiometric formation of butene and Hg(II) from 1 and methane and Hg(II) from methylmercuric chloride eliminates the possibility of an SN1 solvolytic mechanism. As the first well-characterized enzymatic reaction of an organometallic substrate and the first example of an enzyme-mediated SE2 reaction the organomercurial lyase catalyzed carbon-mercury bond cleavage provides an arena for investigating novel enzyme structure-function relationships.     

Interpretation of various ratios of genetic parameters in Hayman's diallelic analysis

The genetical algebra of diallele analysis was used to interpret some relations of genetic parameters which were not earlier applied in research of genetic control (GC). It was discovered that in the case of gene correlation, the relation H2 greater than H1 is possible, meaning the regression among frequencies of alleles in loci. We demonstrated, in what way to distinguish two reasons for changes in H1, H2 and h2, when an external treatment is applied: Due to frequency changes of dominant and recessive alleles in the majority of loci, in case new loci are involved in GC; Due to changes in dominance contributions. The latter could provide new information about a situation in loci by analysing the changes in h2 and the correlation coefficient of parental trait levels with their dominant properties as well as changes in D. A few experimental examples illustrate the theory implication.     

Differential effects of inhibitors on the gamma-secretase complex. Mechanistic implications

Gamma-secretase is a protease complex of four integral membrane proteins, with presenilin (PS) as the apparent catalytic component, and this enzyme processes the transmembrane domains of a variety of substrates, including the amyloid beta-protein precursor and the Notch receptor. Here we explore the mechanisms of structurally diverse gamma-secretase inhibitors by examining their ability to displace an active site-directed photoprobe from PS heterodimers. Most gamma-secretase inhibitors, including a potent inhibitor of the PS-like signal peptide peptidase, blocked the photoprobe from binding to PS1, indicating that these compounds either bind directly to the active site or alter it through an allosteric interaction. Conversely, some reported inhibitors failed to displace this interaction, demonstrating that these compounds do not interfere with the protease by affecting its active site. Differential effects of the inhibitors with respect to photoprobe displacement and in cell-based and cell-free assays suggest that these compounds are important mechanistic tools for deciphering the workings of this intramembrane-cleaving protease complex and its similarity to other polytopic aspartyl proteases.     

Mechanistic analysis of wheat chlorophyllase

Chlorophyllase catalyzes the initial step in the degradation of chlorophyll and plays a key role in leaf senescence and fruit ripening. Here, we report the cloning of chlorophyllase from Triticum aestivum (wheat) and provide a detailed mechanistic analysis of the enzyme. Purification of recombinant chlorophyllase from an Escherichia coli expression system indicates that the enzyme functions as a dimeric protein. Wheat chlorophyllase hydrolyzed the phytol moiety from chlorophyll (k(cat) = 566 min(-1); K(m) = 63 microM) and was active over a broad temperature range (10-75 degrees C). In addition, the enzyme displays carboxylesterase activity toward p-nitrophenyl (PNP)-butyrate, PNP-decanoate, and PNP-palmitate. The pH-dependence of the reaction showed the involvement of an active site residue with a pK(a) of approximately 6.5 for both k(cat) and k(cat)/K(m) with chlorophyll, PNP-butyrate, and PNP-decanoate. Using these substrates, solvent kinetic isotope effects ranging from 1.5 to 1.9 and from 1.4 to 1.9 on k(cat) and k(cat)/K(m), respectively, were observed. Proton inventory experiments suggest the transfer of a single proton in the rate-limiting step. Our analysis of wheat chlorophyllase indicates that the enzyme uses a charge-relay mechanism similar to other carboxylesterases for catalysis. Understanding the activity and mechanism of chlorophyllase provides insight on the biological and chemical control of senescence in plants and lays the groundwork for biotechnological improvement of this enzyme.     

Biotoxicity of lead: influence of various factors

Environmental sources of lead are multiple, and a number of factors influence their toxicity. However, with the exception of tetramethyl and tetraethyl lead, the particular compound of lead seems to have relatively little influence on toxicity compared with the particle size of the source and the route and frequency of exposure. Susceptibility to lead toxicity is greater among immature animals and very young children because of their higher levels of lead ingestion, greater absorption from the gastrointestinal tract, higher percent lead retention in tissues, and greater reactivity of organs, particularly the central nervous system. Marginal nutritional status also increases susceptibility to lead toxicity. Dietary factors influencing toxicity of lead include total calories, calcium, iron, zinc, fat, ascorbic acid, and protein. Although vitamin D, specifically the metabolite 1,25-dihydroxycholecalciferol, increases lead absorption in vitamin D-deficient animals, clinical studies have shown that lead-burdened children have reduced rather than elevated plasma 1,25-dihydroxycholecalciferol levels. Bioavailability of lead, as shown by tissue lead concentrations, is not always an adequate predictor of lead toxicity. For example, concurrent exposure to cadmium results in higher toxicity of lead to the hematopoietic system, but lowers tissue lead levels substantially. Low dietary calcium increases the total body burden of lead but disproportionately increases the deposition of lead in nonosseous tissues.     

The influence of various physical parameters on anther culture of broccoli (Brassica oleracea var. italica)

Embryo formation by cultured broccoli (Brassica oleracea L. var. italica) anthers was best in the pH range of 5.5 to 5.8. Manipulation of the initial medium pH showed, however, that embryos could be recovered throughout the entire pH range tested. Experiments designed to test the influence of anther density on embryo production exhibited an apparent population effect. Comparison of anthers cultured with and without filaments showed a significantly lower level of embryo formation with filaments attached. The importance of anther orientation with the adaxial surface up was also demonstrated. Detailed studies of the effect of temperature on anther response showed the importance of 35°C treatments. Other temperatures and a variety of temperature manipulations were either comparatively ineffective or inhibitory. The duration of 35°C exposure required for optimal response varied widely between 18 and 48 h. Wide variation in plant to plant response was observed despite attempts to optimize the manipulation of physical parameters. Individual plants were identified that reliably formed many thousands of embryos, whereas other plants failed to form embryos under all tested conditions.     

The moderately efficient enzyme: evolutionary and physicochemical trends shaping enzyme parameters

The kinetic parameters of enzymes are key to understanding the rate and specificity of most biological processes. Although specific trends are frequently studied for individual enzymes, global trends are rarely addressed. We performed an analysis of k(cat) and K(M) values of several thousand enzymes collected from the literature. We found that the "average enzyme" exhibits a k(cat) of ~0 s(-1) and a k(cat)/K(M) of ~10(5) s(-1) M(-1), much below the diffusion limit and the characteristic textbook portrayal of kinetically superior enzymes. Why do most enzymes exhibit moderate catalytic efficiencies? Maximal rates may not evolve in cases where weaker selection pressures are expected. We find, for example, that enzymes operating in secondary metabolism are, on average, ~30-fold slower than those of central metabolism. We also find indications that the physicochemical properties of substrates affect the kinetic parameters. Specifically, low molecular mass and hydrophobicity appear to limit K(M) optimization. In accordance, substitution with phosphate, CoA, or other large modifiers considerably lowers the K(M) values of enzymes utilizing the substituted substrates. It therefore appears that both evolutionary selection pressures and physicochemical constraints shape the kinetic parameters of enzymes. It also seems likely that the catalytic efficiency of some enzymes toward their natural substrates could be increased in many cases by natural or laboratory evolution.     

Chitobiase of planktonic crustaceans from South Atlantic coast (Southern Brazil): Characterization and influence of abiotic parameters on enzyme activity

Chitobiase is one of the enzymes involved in chitin degradation in nature. It is produced and released by a variety of organisms from bacteria to fish. In crustaceans, it is associated with digestive function and acts on the epidermis during the molting process. In the present study, the influence of water pH, temperature and salinity on maximum chitobiase activity (MCA), as well as the enzyme affinity (Km) for a substrate, the methylumbelliferyl N-acetyl-ß-d-glucosaminide (MUFNAG) was evaluated in the copepod Acartia tonsa. Km values for chitobiases of other crustaceans from the Patos Lagoon estuary and Cassino Beach (Southern Brazil) were also determined. For A. tonsa, MCA was observed at pH 5-6 and 30-35 °C. The range of pH was quite similar to that reported for other aquatic organisms. However, the range of temperature was lower than that previously reported. For salinity, no previous studies have considered the influence of this parameter on MCA. For A. tonsa, MCA was observed in freshwater, showing a significant linear decrease with increasing salinity. Considering that maximum copepod survival and growth rates are observed between 15 and 25 ppt, these findings suggest that the observed enzyme activity in this range of salinity (68 to 47% of that measured in freshwater) is not a limiting factor for A. tonsa growth. However, the extremely decreased enzyme activity observed in salinity 30 ppt (33% of that measured in freshwater) suggests that chitobiase activity might be one of the limiting factor for copepod growth at 30 ppt salinity or higher. Km values (μM) determined for organisms evaluated in the present study (copepod A. tonsa = 20.77; mysid Metamysidopsis elongata atlantica = 14.67; nauplii barnacle Balanus improvisus = 18.19; decapod zoea = 14.30; decapod megalopa = 24.77) were lower than those reported for other crustaceans from Northern Hemisphere. Also, they were much lower than those of organisms from different taxonomic groups like bacteria and fungi, but much higher than in protozoans and dinoflagelates. These findings suggest that chitobiase might be differentially evolved in each specific group of organism, and even within different ontogenetic stages of the same species, for a better adaptation to cope with its respective environmental needs.