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PubMed1993

Ribonucleotide reductases and their occurrence in microorganisms: a link to the RNA/DNA transition.

The evolution of a deoxyribonucleotide synthesizing ribonucleotide reductase might have initiated the transition from the ancient RNA world into the prevailing DNA world. At least five classes of ribonucleotide reductases have evolved. The ancient enzyme has not been identified. A reconstruction of the first ribonucleotide reductase requires knowledge of contemporary enzymes and of microbial evolution. Experimental work on the former focuses on few organisms, whereas the latter is now well understood on the basis of ribosomal RNA sequences. Deoxyribonucleotide formation has not been investigated in many evolutionary important microorganisms. This review covers our knowledge on deoxyribonucleotide synthesis in microorganisms and the distribution of ribonucleotide reductases in nature. Ecological constraints on enzyme evolution and knowledge deficiencies emerge from complete coverage of the phylogenetic groups.

Animals↗

PubMed2003

A perspective on enzyme catalysis.

The seminal hypotheses proposed over the years for enzymatic catalysis are scrutinized. The historical record is explored from both biochemical and theoretical perspectives. Particular attention is given to the impact of molecular motions within the protein on the enzyme's catalytic properties. A case study for the enzyme dihydrofolate reductase provides evidence for coupled networks of predominantly conserved residues that influence the protein structure and motion. Such coupled networks have important implications for the origin and evolution of enzymes, as well as for protein engineering.

Binding Sites↗

PubMed1984

Eight new restriction endonucleases fröm Herpetosiphon giganteus--divergent evolution in a family of enzymes.

Characterization of eight restriction endonucleases isolated from five strains of Herpetosiphon giganteus is described. HgiCI from strain Hpg9 recognizes and cleaves the degenerate sequence: GGPyPuCC, producing 5'-hexanucleotide protruding ends. Endonucleases HgiBI, HgiCII and HgiEI are isoschizomers of AvaII; HgiCIII and HgiDII are isoschizomers of SalI; and HgiDI and HgiGI are isoschizomers of AcyI. Based upon their closely related and in part overlapping recognition specificities a close evolutionary relationship is proposed for all known Hgi restriction endonucleases.

Base Sequence↗

PubMed1998

Computer simulations of enzyme catalysis: finding out what has been optimized by evolution.

The origin of the catalytic power of enzymes is discussed, paying attention to evolutionary constraints. It is pointed out that enzyme catalysis reflects energy contributions that cannot be determined uniquely by current experimental approaches without augmenting the analysis by computer simulation studies. The use of energy considerations and computer simulations allows one to exclude many of the popular proposals for the way enzymes work. It appears that the standard approaches used by organic chemists to catalyze reactions in solutions are not used by enzymes. This point is illustrated by considering the desolvation hypothesis and showing that it cannot account for a large increase in kcat relative to the corresponding kcage for the reference reaction in a solvent cage. The problems associated with other frequently invoked mechanisms also are outlined. Furthermore, it is pointed out that mutation studies are inconsistent with ground state destabilization mechanisms. After considering factors that were not optimized by evolution, we review computer simulation studies that reproduced the overall catalytic effect of different enzymes. These studies pointed toward electrostatic effects as the most important catalytic contributions. The nature of this electrostatic stabilization mechanism is far from being obvious because the electrostatic interaction between the reacting system and the surrounding area is similar in enzymes and in solution. However, the difference is that enzymes have a preorganized dipolar environment that does not have to pay the reorganization energy for stabilizing the relevant transition states. Apparently, the catalytic power of enzymes is stored in their folding energy in the form of the preorganized polar environment.

Animals↗

PubMed2007

The dependence of enzyme activity on temperature: determination and validation of parameters.

Traditionally, the dependence of enzyme activity on temperature has been described by a model consisting of two processes: the catalytic reaction defined by DeltaG(Dagger)(cat), and irreversible inactivation defined by DeltaG(Dagger)(inact). However, such a model does not account for the observed temperature-dependent behaviour of enzymes, and a new model has been developed and validated. This model (the Equilibrium Model) describes a new mechanism by which enzymes lose activity at high temperatures, by including an inactive form of the enzyme (E(inact)) that is in reversible equilibrium with the active form (E(act)); it is the inactive form that undergoes irreversible thermal inactivation to the thermally denatured state. This equilibrium is described by an equilibrium constant whose temperature-dependence is characterized in terms of the enthalpy of the equilibrium, DeltaH(eq), and a new thermal parameter, T(eq), which is the temperature at which the concentrations of E(act) and E(inact) are equal; T(eq) may therefore be regarded as the thermal equivalent of K(m). Characterization of an enzyme with respect to its temperature-dependent behaviour must therefore include a determination of these intrinsic properties. The Equilibrium Model has major implications for enzymology, biotechnology and understanding the evolution of enzymes. The present study presents a new direct data-fitting method based on fitting progress curves directly to the Equilibrium Model, and assesses the robustness of this procedure and the effect of assay data on the accurate determination of T(eq) and its associated parameters. It also describes simpler experimental methods for their determination than have been previously available, including those required for the application of the Equilibrium Model to non-ideal enzyme reactions.

Acid Phosphatase↗

PubMed2003

Retention of enzyme gene duplicates by subfunctionalization.

Duplication-degeneration-complementation (DDC) describes a process by which evolving duplicates of a pleiotropic ancestral gene divide up the multiple functions of the ancestor between them (i.e. subfunctionalize), and this ultimately frustrates the rate of pseudogene formation. Focusing explicitly on enzyme-like pleiotropic function, we model DDC driven by sequence divergence between duplicates. The model incorporates an idealized sequence-function mapping in which enzyme-substrate binding affinity is related to hydrophobic versus polar (HP) amino-acid composition of tertiary structure about the binding pocket. In this sense, a transparent coupling between physical-chemical function of an enzyme and sequence evolution is presented.

Enzymes↗

PubMed2002

Origin and evolution of arthropod hemocyanins and related proteins.

Arthropod hemocyanins are large, multimeric, (n x 6) copper-containing proteins that deliver oxygen in the haemolymph of many chelicerate, crustacean, myriapod, and also possibly some insect species. The arthropod hemocyanins belong to a large protein superfamily that also includes the arthropod phenoloxidases, certain crustacean and insect storage proteins (pseudo-hemocyanins and hexamerins), and the insect hexamerin receptors. Here I summarise the present knowledge of the origin, functional adaptations, and evolution of these proteins. Arthropod and mollusc hemocyanins are, if at all, only distantly related. As early as in the arthropod stem line, the hemocyanins emerged from a phenoloxidase-like enzyme. The evolution of distinct hemocyanin subunits, as well as the formation of multi-hexamers occurred independently within the arthropod subphyla. Hemocyanin subunit evolution is strikingly different in the Chelicerata, Myriapoda and Crustacea. Hemocyanins individually gave rise to two distinct copper-less storage proteins, the insect hexamerins and the crustacean pseudo-hemocyanins (cryptocyanins). The receptor responsible for the uptake of hexamerin by the larval fat body of the insects emerged from a hexamerin-precursor. Molecular phylogenetic analyses show a close relationship of the crustacean and insect proteins, providing strong support for a pancrustacean taxon, while structural data suggest a myriapod-chelicerate clade.

Animals↗

PubMed1982

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.

Biological Evolution↗

PubMed2000

Genetics and the evolutionary process.

Population genetics was put forward as a mathematical theory between 1918 and 1932 and played a leading part in the rediscovery of the concept of natural selection. As an autonomous science developing Mendel's laws at the population scale and a key element of the Darwinian theory of evolution, its dual status led its practioners to initially overlook some consequences of Mendelism not accounted for by the Darwinian theory, including random drift and the cost of selection. The latter were put forward on purely theoretical grounds in the 1950s, but their importance was acknowledged only when empirical data on protein evolution and enzyme polymorphism (since 1965) and on DNA variation (since 1983) were obtained. The neutralist/selectionist debate that ensued involved disagreement over the scientific method as well as over the mechanisms of molecular evolution. Population genetics has long assumed the existence of natural selection a priori. It has since recentred around the null hypothesis that molecular evolution is neutral. This new approach, applied to sequence comparison and to the study of linkage disequilibrium, is logically more justified, yet empirical observations derived from it paradoxically show the overwhelming importance of selective effects within genomes.

Animals↗

PubMed1977

Relationship between enzyme heterozygosity and quaternary structure.

The need for proteins to maintain particular quarternary structures constrains variability in amino acid sequence. Monomeric enzymes are then expected to be more variable than dimeric forms, which in turn are expected to be more variable than tetrameric forms. These predictions are confirmed by analysis of available data on enzyme variation. Theories relating enzyme heterozygosity to metabolic function are discussed in the light of these findings.

Amino Acid Sequence↗

PubMed1991

Why do many Michaelian enzymes exhibit an equilibrium constant close to unity for the interconversion of enzyme-bound substrate and product?

1. The idea is advanced that an evolutionary pressure in the direction of increased metabolic fluxes should lead to the selection at any evolutionary stage of enzymes which are optimally efficient considering the total evolutionary effort which has been spent on their improvement as catalysts. Relationships are derived which may be used for determination of the optimal state of operation of a Michaelian enzyme at a given total evolutionary effort as measured, for instance, by the mean magnitude of variable rate constants in the reaction mechanism. These relationships are applied to characterize the dependence on the total evolutionary effort of the optimal value of the equilibrium constant (Kint) for the conversion of enzyme-bound substrate into enzyme-bound product. 2. The results show that the optimal value of Kint reflects primarily the magnitude of the overall equilibrium constant for the catalysed reaction, whether or not the enzyme operates close to equilibrium. When the total evolutionary effort tends towards infinitely high values, Kint approaches unity, regardless of the magnitude of the overall equilibrium constant and of the concentrations of substrate and product. Concomitantly, the enzyme approaches its ultimate state of catalytic perfection where all variable rate constants in the mechanism tend towards infinitely high values. 3. It is concluded from previously reported kinetic data that triosephosphate isomerase has been subjected to such a low total evolutionary effort that the enzyme cannot possibly have reached (or even significantly approached) the ultimate state of perfection where the Kint value necessarily becomes close to unity. The reason why this enzyme exhibits a Kint value relatively close to unity is that it catalyses a reaction with an equilibrium constant relatively close to unity. Evidence is presented to show that similar considerations may apply for other enzymes operating by the examined reaction mechanism. 4. Analytical data are reported which indicate that Kint ultimately approaches unity because forward and reverse rate constants for the interconversion of enzyme-bound substrate and product are equivalent with regard to the evolutionary effort required to increase their magnitude.

Biological Evolution↗

PubMedDate not supplied

[Enzymatic mechanisms of compensation of deleterious mutations].

Mechanisms of stabilization and compensation, that occur in biochemical systems with enzymes modified by harmful mutations are considered. The compensation of such mutations can result in their evolutionary neutralism. The stabilization is considered due to kinetic signals of metabolites which form the direct and feedback connections with enzymes (temporal stabilization), and also the compensation in enzymatic aggregates determined by the changes of conformation (spatial stabilization). Examples of the stabilization in one or several steady states of enzymatic systems are presented. The neutralism of the distortion of inhibitory and catalytic properties of enzymes is shown in the region of stabilization of these properties.

Animals↗

PubMed1976

Properties and function of clostridial membrane ATPase.

ATPase (ATP phosphohydrolase, EC 3.6.1.3) was detected in the membrane fraction of the strict anaerobic bacterium, Clostridium pasteurianum. About 70% of the total activity was found in the particulate fraction. The enzyme was Mg2+ dependent; Co2+ and Mn2+ but not Ca2+ could replace Mg2+ to some extent; the activation by Mg2+ was slightly antagonized by Ca2+. Even in the presence of Mg2+, Na+ or K+ had no stimulatory effect. The ATPase reaction was effectively inhibited by one of its products, ADP, and only slightly by the other product, inorganic phosphate. Of the nucleoside triphosphates tested ATP was hydrolyzed with highest affinity ([S]0.5 v = 1.3 mM) and maximal activity (120 U/g). The ATPase activity could be nearly completely solubilized by treatment of the membranes with 2 M LiCl in the absence of Mg2+. Solubilization, however, led to instability of the enzyme. The clostridial solubilized and membrane-bound ATPase showed different properties similar to the "allotopic" properties of mitochondrial and other bacterial ATPases. The membrane-bound ATPase in contrast to the soluble ATPase was sensitive to the ATPase inhibitor dicyclohexylcarbodiimide (DCCD). DCCD, at 10(-4) M, led to 80% inhibition of the membrane-bound enzyme; oligomycin ouabain, or NaN3 had no effect. The membrane-bound ATPase could not be stimulated by trypsin pretreatment. Since none of the mono- or divalent cations had any truly stimulatory effect, and since a pH gradient (interior alkaline), which was sensitive to the ATPase inhibitor DCCD, was maintained during growth of C. pasteurianum, it was concluded that the function of the clostridial ATPase was the same as that of the rather similar mitochondrial enzyme, namely H+ translocation. A H+-translocating, ATP-consuming ATPase appears to be intrinsic equipment of all prolaryotic cells and as such to be phylogenetically very old; in the course of evolution the enzyme might have been developed to a H+-(re)translocating, ATP-forming ATPase as probably realized in aerobic bacteria, mitochondria and chloroplasts.

2,6-Dichloroindophenol↗

PubMed2003

Evolution of function in (beta/alpha)8-barrel enzymes.

The (beta/alpha)(8)-barrel is the most common fold in structurally characterized enzymes. Whether the functionally diverse enzymes that share this fold are the products of either divergent or convergent evolution (or both) is an unresolved question that will probably be answered as the sequence databases continue to expand. Recent work has examined natural, designed, and directed evolution of function in several superfamilies of (beta/alpha)(8)-barrel containing enzymes.

Animals↗

PubMed1992

Immunocytochemical and biochemical demonstration of formaldhyde dehydrogenase (class III alcohol dehydrogenase) in the nucleus.

Alcohol dehydrogenase (ADH), the major enzyme catalyzing the biological oxidation of ethanol in mammals, includes four classes with very different capacities for ethanol oxidation. Class III ADH is present in all the tissues and is well conserved throughout evolution. This enzyme has a low activity with ethanol, is specific for the glutathione-dependent oxidation of formaldehyde, and is therefore a formaldehyde dehydrogenase (FALDH). Until now there have been few and conflicting studies concerning its intracellular distribution, which is important for the understanding of its role in cell function. In the present work we used biochemical and immunocytochemical methods to assess the distribution of FALDH in rat hepatocytes and astroglial cells. With the glutathione-dependent formaldehyde dehydrogenase assay, we found the highest activity in the cytosol of hepatocytes and brain cells (12 and 2.6 mU/mg protein, respectively), but nuclei also exhibited significant activity (1.16 and 2.1 mU/mg protein, respectively). The immunocytochemical results showed the presence of FALDH binding sites in both the cytoplasm and the nucleus of the different cell types studied. Whereas no specific gold particle labeling was seen associated with any cytoplasmic component, in the nucleus the particles were found mainly over condensed chromatin and interchromatin regions. Finally, the gold particle density over both the nucleus and cytoplasm was greater in differentiated than in proliferating astrocytes in primary culture. In contrast, class I ADH, primarily responsible for ethanol metabolism, was found only in the cytoplasm of hepatocytes. We propose that one of the functions of FALDH is to protect cell structures, including DNA, from the toxic effects of endogenous formaldehyde, which is an intermediate in many metabolic process.

Aldehyde Oxidoreductases↗

PubMed1985

Gene regulatory divergence among species estimated by altered developmental patterns in interspecific hybrids.

Disturbances in the schedules of gene expression in developing interspecific fish hybrids have been used to draw inferences about the extent of gene regulatory divergence between species and about the degree to which this gene regulatory divergence is correlated with structural gene divergence, as estimated by genetic distance. Sperm from each of 10 different species representing six genera within the family Centrarchidae was used to fertilize eggs of the Florida largemouth bass (Micropterus salmoides floridanus). The genetic distances (D; Nei 1978) between the parental species used to form the hybrids ranged from 0.133 to 0.974. The developmental success and temporal patterns of gene expression of each of the hybrids were compared with those of the Florida largemouth bass. As the genetic distance between the paternal species and the Florida largemouth bass increased, there was a general decline in developmental success in the hybrid embryos as demonstrated by the observed reductions in the percentage of hatching and by progressively earlier and more extensive morphological abnormalities. Concomitantly, progressively more marked alterations in developmental schedules of expression of 15 enzyme loci occurred in the hybrids as the genetic distance between parental species increased. However, observed deviations from this trend for a few species may represent an uncoupling of the rates and modes of evolution of structural genes from those for genes regulating developmental processes.

Animals↗

PubMed2006

Arsenate reduction: thiol cascade chemistry with convergent evolution.

The frequent abundance of arsenic in the environment has guided the evolution of enzymes for the reduction of arsenate. The arsenate reductases (ArsC) from different sources have unrelated sequences and structural folds, and can be divided into different classes on the basis of their structures, reduction mechanisms and the locations of catalytic cysteine residues. The thioredoxin-coupled arsenate reductase class is represented by Staphylococcus aureus pI258 ArsC and Bacillus subtilis ArsC. The ArsC from Escherichia coli plasmid R773 and the eukaryotic ACR2p reductase from Saccharomyces cerevisiae represent two distinct glutaredoxin-linked ArsC classes. All are small cytoplasmic redox enzymes that reduce arsenate to arsenite by the sequential involvement of three different thiolate nucleophiles that function as a redox cascade. In contrast, the ArrAB complex is a bacterial heterodimeric periplasmic or a surface-anchored arsenate reductase that functions as a terminal electron acceptor and transfers electrons from the membrane respiratory chain to arsenate. Finally, the less well documented arsenate reductase activity of the monomeric arsenic(III) methylase, which is an S-adenosylmethionine (AdoMet)-dependent methyltransferase. After each oxidative methylation cycle and before the next methylation step, As(V) is reduced to As(III). Methylation by this enzyme is also considered an arsenic-resistance mechanism for bacteria, fungi and mammals.

Animals↗

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↗