The perfection of substrate-channelling in interacting enzyme systems: energetics and evolution

Some implications of substrate channelling in interacting enzyme systems are considered, with regard to the energetics and evolution of enzyme action. The transient time, a key analytical parameter relating to the phenomenon of channelling, is the basis of our kinetic study. Bounds on the kinetics of multienzyme complexes are established using (apparent) rate constants emanating from the transient-time formulation of coupled reactions. From a transition state representation of the rate process, it is shown how dynamically and statically organized enzyme systems lead to the modification of current ideas on the evolutionary optimization of the energy profile of enzyme catalysis in situ.     

Studies on the effects of structure on the behavior of enzyme systems

The effects of structural features on various properties of enzyme systems are studied. Some of the effects are: in a homogeneous reaction, enzyme compartmentalization decreases the rate; in a heterogeneous reaction, compartmentalization increases the rate. The steady-state concentration of intermediates is larger in a non-uniform than in uniform systems. Periodicities do not generally occur in the common kinetic systems; they do occur in autocatalytic systems, but compartmentalization reduces their probability of occurrence. The conditions for overshoot are different for uniform and non-uniform systems. Multiple stable steady states are not a common occurrence among biologically typical reactions; they do occur in combined autocatalytic and surface systems (a mechanism for the gener position effect is suggested by this property). The local pH is affected by the enzyme aggregation as well as by the geometry of the enzyme structure. A 2-step system can give rise to the characteristic rate vs. pH curve, where the optimum is not necessarily at isoelectric point. The expression for the osmotic pressure inside a spherical particle is deduced. The pressure is shown to be dependent on the radius. The rate inside a cell particle is shown to be determined by the shape of the particle.     

Regulation and degradation of HMGCo-A reductase

The enzyme, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) controls the biosynthesis of cholesterol. Hypercholesterolemia and atherosclerosis are critical health risk factors. One way of controlling these risk factors is to manipulate regulation as well as degradation of HMGR. At present, a class of compounds called statins, which are HMGR inhibitors, are used for the treatment of hypercholesterolemia. However, statins suffer major setbacks as their use produces more adverse reactions than the desirable one of inhibiting the enzyme. Genetically engineered forms of HMGR are also studied in primitive life forms like bacteria, but detailed investigation of this enzyme in human systems is certainly required. Extensive studies have been made on the regulatory aspects of this enzyme, but no breakthrough is conspicuous in the clinical background to find an alternative treatment for hypercholesterolemia. The immediate need is to find an alternate way of regulating degradation of the enzyme. This review presents the importance of regulation and degradation of the HMGR enzyme in different systems to gain possible insight into alternative schemes for regulating this enzyme and, if these exist, the feasibility of extending them same to studies in mammalian systems. A high degree of similarity exists between mammalian and yeast HMGR. Detailed studies reported on the regulation and degradation of the yeast enzyme also throw more light on the mammalian system, leading to a better understanding of ways of controlling hypercholesterolemia.     

Using enzyme cascades in biocatalysis: Highlight on transaminases and carboxylic acid reductases

Biocatalysis, the use of enzymes in chemical transformations, is an important green chemistry tool. Cascade reactions combine different enzyme activities in a sequential set of reactions. Cascades can occur within a living (usually bacterial) cell; in vitro in ‘one pot’ systems where the desired enzymes are mixed together to carry out the multi-enzyme reaction; or using microfluidic systems. Microfluidics offers particular advantages when the product of the reaction inhibits the enzyme(s). In vitro systems allow variation of different enzyme concentrations to optimise the metabolic ‘flux’, and the addition of enzyme cofactors as required. Cascades including cofactor recycling systems and modelling approaches are being developed to optimise cascades for wider industrial scale use. Two industrially important enzymes, transaminases and carboxylic acid reductases are used as examples regarding their applications in cascade reactions with other enzyme classes to obtain important synthons of pharmaceutical interest.     

Enzyme stabilization by nano/microsized hybrid materials

Immobilization is a key technology for successful realization of enzyme‐based industrial processes, particularly for production of green and sustainable energy or chemicals from biomass‐derived catalytic conversion. Different methods to immobilize enzymes are critically reviewed. In principle, enzymes are immobilized via three major routes (i) binding to a support, (ii) encapsulation or entrapment, or (iii) cross‐linking (carrier free). As a result, immobilizing enzymes on certain supports can enhance storage and operational stability. In addition, recent breakthroughs in nano and hybrid technology have made various materials more affordable hosts for enzyme immobilization. This review discusses different approaches to improve enzyme stability in various materials such as nanoparticles, nanofibers, mesoporous materials, sol–gel silica, and alginate‐based microspheres. The advantages of stabilized enzyme systems are from its simple separation and ease recovery for reuse, while maintaining activity and selectivity. This review also considers the latest studies conducted on different enzymes immobilized on various support materials with immense potential for biosensor, antibiotic production, food industry, biodiesel production, and bioremediation, because stabilized enzyme systems are expected to be environmental friendly, inexpensive, and easy to use for enzyme‐based industrial applications.     

Mean Lifetime and First-Passage Time of the Enzyme Species Involved in an Enzyme Reaction. Application to Unstable Enzyme Systems

Taking as starting point the complete analysis of mean residence times in linear compartmental systems performed by Garcia-Meseguer et al. (Bull. Math. Biol. 65:279–308, 2003) as well as the fact that enzyme systems, in which the interconversions between the different enzyme species involved are of first or pseudofirst order, act as linear compartmental systems, we hereby carry out a complete analysis of the mean lifetime that the enzyme molecules spend as part of the enzyme species, forms, or groups involved in an enzyme reaction mechanism. The formulas to evaluate these times are given as a function of the individual rate constants and the initial concentrations of the involved species at the onset of the reaction. We apply the results to unstable enzyme systems and support the results by using a concrete example of such systems. The practicality of obtaining the mean times and their possible application in a kinetic data analysis is discussed.     

Catalase Stability in Amorphous and Supercooled Media Related to Trehalose- Water- Salt Interactions

Sugars are the most common excipients added to pharmaceutical and biotechnological formulations as protein protectants due to their adequate physicochemical properties, low toxicity, high purity and low cost. However, a second excipient is generally required to extend their protection in supercooled media. The effect of electrolytes is of special interest because of their universal presence in biological systems, their major influence on water structure, and their capability to interact with biomolecules. The purpose of the present work was to analyze the effect of different salts on the stability of catalase in amorphous (glassy and supercooled) trehalose matrices. Trehalose-Mg²⁺ system was better than trehalose alone to protect the enzyme both during freeze-drying and later storage at low RH (22%). The stabilizing effect observed for certain salts in these systems was not related with an increase of the T_g value. Under conditions at which trehalose crystallizes (43 %RH), salts (especially Mg²⁺) were detrimental since the enzyme became confined in a salt-concentrated region. Protein denaturation and aggregation were analyzed through differential scanning calorimetry in order to correlate activity changes with physical changes. Trehalose systems without salt and Mg²⁺-containing systems showed almost no aggregation after denaturation, in agreement with the thermal stability of the enzyme. Thus, the two major parameters related to enzyme stability in freeze-dried non-crystallized systems are: enzyme characteristics (type, quaternary structure) and salt-protein specific interactions.

     

Microbial xylanases and their industrial applications: a review

Despite an increased knowledge of microbial xylanolytic systems in the past few years, further studies are required to achieve a complete understanding of the mechanism of xylan degradation by microorganisms and their enzymes. The enzyme system used by microbes for the metabolism of xylan is the most important tool for investigating the use of the second most abundant polysaccharide (xylan) in nature. Recent studies on microbial xylanolytic systems have generally focussed on induction of enzyme production under different conditions, purification, characterization, molecular cloning and expression, and use of enzyme predominantly for pulp bleaching. Rationale approaches to achieve these goals require a detailed knowledge of the regulatory mechanism governing enzyme production. This review will focus on complex xylan structure and the microbial enzyme complex involved in its complete breakdown, studies on xylanase regulation and production and their potential industrial applications, with special reference to biobleaching.     

Modulating enzyme activity using ionic liquids or surfactants

One of the important strategies for modulating enzyme activity is the use of additives to affect their microenvironment and subsequently make them suitable for use in different industrial processes. Ionic liquids (ILs) have been investigated extensively in recent years as such additives. They are a class of solvents with peculiar properties and a "green" reputation in comparison to classical organic solvents. ILs as co-solvents in aqueous systems have an effect on substrate solubility, enzyme structure and on enzyme–water interactions. These effects can lead to higher reaction yields, improved selectivity, and changes in substrate specificity, and thus there is great potential for IL incorporation in biocatalysis. The use of surfactants, which are usually denaturating agents, as additives in enzymatic reactions is less reviewed in recent years. However, interesting modulations in enzyme activity in their presence have been reported. In the case of surfactants there is a more pronounced effect on the enzyme structure, as can be observed in a number of crystal structures obtained in their presence. For each additive and enzymatic process, a specific optimization process is needed and there is no one-fits-all solution. Combining ILs and surfactants in either mixed micelles or water-in-IL microemulsions for use in enzymatic reaction systems is a promising direction which may further expand the range of enzyme applications in industrial processes. While many reviews exist on the use of ILs in biocatalysis, the present review centers on systems in which ILs or surfactants were able to modulate and improve the natural activity of enzymes in aqueous systems.     

Ionic strength: A neglected variable in enzyme technology

Ionic strength is a variable of considerable importance in enzyme biochemistry. Unfortunately, the ionic strength variable is little used in immobilized enzyme studies. This paper gives examples of enzyme systems known to be sensitive to ionic strength. Suggestions are offered for the manipulation of ionic strength in enzyme technology studies. Laboratory results on the effect of ionic strength on the reaction catalysed by immobilized pancreatic ribonuclease (EC 3.1.27.5) are also presented and analysed using the Debye-Hückel equation.     

Dynamics of non-linear biochemical systems and the evolutionary significance of time hierarchy

Several non-linear reaction networks are analyzed in order to study the influence of time hierarchy on the dynamics of biochemical systems. The analysis is based on the assumption that the separation of the time constants within metabolic systems is a direct consequence of strong differences of enzyme concentrations. Therefore, as variable system parameters, only the enzyme concentrations are considered. By investigation of the stationary states of various three-component models with feedback-activation bifurcation diagrams within a two-dimensional parameter space, the enzyme simplex, are constructed. The diagrams contain the information about the number of stationary states, their stability properties as well as the type of motion expected at different parameter combinations. The results support the hypothesis that systems with separated time constants generally show a simple dynamic behaviour. Complex motions can be expected mainly for systems without time hierarchy, which are characterized by parameters located within the centre of the enzyme simplex. Quantitative measures for the time hierarchy and the complexity of the dynamics are derived. It is supposed that the separation of time constants is a main feature of the evolution of biochemical systems. The hypothesis is further supported by the consideration of the time hierarchy of the glycolytic pathway.     

Enzyme diffusion and action on soluble and insoluble substrate biopolymers

The action of enzymes on soluble and insoluble substrate biopolymers is discussed, taking into account enzyme diffusion along the biopolymer “surface” and interaction with interspersed ligand groups that may be modified by the action of the enzyme. It is shown that movement of the enzyme under trhe combined effect of these two processes can be described as a diffusion process characterized by an apparent diffusion coefficient that generally depends on both time and position. Equations describing the system are formulated and some specific examples analyzed in terms of analytical or numerical solutions. The concentration distributions of both the enzyme and the substrate (or product ) were obtained for different systems for which the apparent diffusion coefficient is a function of time only, as well as of both time and position. The relevance of the formulation, as developed, to systems in which reduction in dimensionality leads to enhanced enzyme efficiency is discussed, and possible uses of the theory in studies of biopolymer structure and enzyme-biopolymer interactions are suggested.     

Red cell antigen, serum protein and red cell enzyme polymorphisms in Karkar Islanders and inhabitants of the adjacent North Coast of New Guinea

Blood samples from the Waskia and Takia populations of Karkar Island, Papua New Guinea, and other nearby mainland populations, were tested for genetic variation in blood group, serum protein and red cell enzyme systems. Polymorphic variation was present in the ABO, P, MNS, Rh, Lewis, Duffy, Kidd and Gerbich blood group systems, in the Hp and Tf serum protein systems, and in the acid phosphatase, 6-PGD, ADA, PGM, MDH, and G-6-PD enzyme systems. A small number of variants was found in other systems: there were 4 Lu(a+), 1 Kp(a+), 2 C variants in the acid phosphatase system, 6 LDH variants, 1 ADA3-1 and 1 AK2-1 sample. All samples were negative for the red cell antigens Cw, Vw, He, K, Jsa, Dia, Wra, Rd and Marriott, and no variation was observed in the PHI enzyme system. The results are discussed in relation to those obtained on other Papua New Guinea populations.     

Thermal stability of membrane-reconstituted yeast cytochrome c oxidase

The thermal dependence of the structural stability of membrane-reconstituted yeast cytochrome c oxidase has been studied by using different techniques including high-sensitivity differential scanning calorimetry, differential detergent solubility thermal gel analysis, and enzyme activity measurements. For these studies, the enzyme has been reconstituted into dimyristoylphosphatidylcholine (DMPC) and dielaidoylphosphatidylcholine (DEPC) vesicles using detergent dialysis. The phospholipid moiety affects the stability of the enzyme as judged by the dependence of the denaturation temperature on the lipid composition of the bilayer. The enzyme is more stable when reconstituted with the 18-carbon, unsaturated phospholipid (DEPC) than with the 14-carbon saturated phospholipid (DMPC). In addition, the shapes of the calorimetric transition profiles are different in the two lipid systems, indicating that not all of the subunits are affected equally by the lipid moiety. The overall enthalpy change for the enzyme denaturation is essentially the same for the two lipid reconstitutions (405 kcal/mol of protein for the DMPC and 425 kcal/mol for the DEPC-reconstituted enzyme). In both systems, the van't Hoff to calorimetric enthalpy ratios are less than 0.2, indicating that the unfolding of the enzyme cannot be represented as a two-state process. Differential detergent solubility experiments have allowed us to determine individual subunit thermal denaturation profiles. These experiments indicate that the major contributors to the main transition peak observed calorimetrically are subunits I and II and that the transition temperature of subunit III is the most affected by the phospholipid moiety. Experiments performed at different scanning rates indicate that the thermal denaturation of the enzyme is a kinetically controlled process characterized by activation energies on the order of 40 kcal/mol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolites and pathway flexibility

Flexibility of metabolites and enzymes is investigated (i) on the level of the individual molecule, (ii) on the pathway level and (iii) combined effects on the systems and network level. Tools and results from our current research are summarized including data from our metabolite enzyme database. Including our latest census we find frequently used metabolites stimulate evolutionary flexibility in specific enzyme superfamilies. Furthermore, simultaneous changes of reactions and metabolites are observed in these flexible enzyme superfamilies. Both effects provide a strong source for resistance in parasites and pathogens. Specific adaptations scenarios and some counter strategies are discussed.     

A three-dimensional solubility parameter approach to nonaqueous enzymology

Widespread commercial application of enzymes as catalysts for specialty or commodity chemical synthesis will require their use in nonaqueous systems. While a number of non-aqueous enzyme applications have been demonstrated, the lack of useful rules for predicting enzyme-solvent interactions has hindered the development of this technology. Both Hildebrand and solvent hydrophobicity (octanol-water partition coefficient) parameters have been used previously to correlate and predict enzyme activity in nonaqueous systems, with some success, but any single-parameter approach is inherently limited in its ability to reflect the spectrum of possible enzyme-solvent interactions. Therefore, this study evaluates the three-dimensional solubility parameter space, as proposed by Hansen, to correlate and predict enzyme activity in microaqueous, miscible, and biphasic nonaqueous systems. Preliminary results suggest that Hansen parameters may be useful for correlating nonaqueous enzyme activity, and that the dispersive and polar parameters may be disproportionately important in single-phase microaqueous systems. The Hansen hydrogen-bonding parameter appears to be the only parameter yet evaluated capable of correlating the water requirement for enzyme activity in microaqueous systems, suggesting that water affects protein structure through enthalpic rather than entropic processes in nonaqueous systems. Insufficient data are available for miscible and biphasic systems, but it is proposed that enzyme activity may correlate with the average solubility parameters of miscible systems and of the aqueous phase in biphasic systems.     

Simple Biochemical Pathways far from Steady State Can Provide Switchlike and Integrated Responses

Covalent modification cycles (systems in which the activity of a substrate is regulated by the action of two opposing enzymes) and ligand/receptor interactions are ubiquitous in signaling systems and their steady-state properties are well understood. However, the behavior of such systems far from steady state remains unclear. Here, we analyze the properties of covalent modification cycles and ligand/receptor interactions driven by the accumulation of the activating enzyme and the ligand, respectively. We show that for a large range of parameters both systems produce sharp switchlike response and yet allow for temporal integration of the signal, two desirable signaling properties. Ultrasensitivity is observed also in a region of parameters where the steady-state response is hyperbolic. The temporal integration properties are tunable by regulating the levels of the deactivating enzyme and receptor, as well as by adjusting the rate of accumulation of the activating enzyme and ligand. We propose that this tunability is used to generate precise responses in signaling systems.     

Prevention of Aging. Communication I. Individual Enzymatic Variations of the Antioxidant System and a Way to Correct this System by Means of Electrochemically Activated Systems

The effect of electrochemically activated systems (ECAS) on the enzyme activity of the antioxidant system (catalase, peroxidase, and superoxide dismutase) appears to be a normalizing effect that increases supressed enzyme activity and decreases enhanced enzyme activity. Baseline enzyme activity is characterized by significant individual variations in both animals and humans. The effect of ECAS may be explained by the training effect of electroactivated systems due to the excess of electrons, with the ECAS redox potential being negative.