Double-site enzymes and squatting. A study of the regulation by one or several ligands binding at two different classes of site

The existence of two (generally different) binding sites per protomer for the same ligand has already been observed (“double-site enzymes”). Furthermore, in some instances, the two sites appear to be shared in common by two or more ligands (“squatting”). The theoretical implications of such cases have been emphasized here using a generalization of an allosteric V model. This model, by taking into account the competition between ligands which occurs in vivo, evidences various, or even peculiar regulatory patterns, and could be of general physiological interest. It has been illustrated here by some real systems.     

Substrate-containing gel electrophoresis: sensitive detection of amylolytic, nucleolytic, and proteolytic enzymes

Electrophoresis of hydrolytic enzymes under nondenaturing conditions on acrylamide gels containing the appropriate high-molecular-weight substrates entrapped on the gel has been explored as a general method for sensitive enzyme resolution and detection. Under electrophoresis conditions of optimal enzyme activity, the enzymes may bind tightly to the fixed substrate and can only migrate in the electrophoretic field as the substrate is hydrolyzed. When the gels after electrophoresis in this “binding mode” are stained with substrate-detecting reagents, clear tracks of enzyme migration are observed, and the length of each track is a function of the amount of enzyme present in that track. Multiple forms of a given enzyme activity have not been and are not likely to be observed under these conditions. Under electrophoresis conditions of minimal (or suboptimal) enzyme activity, the enzymes do not bind to the fixed substrate and their mobility in the electrophoretic field does not appear to be significantly affected by the presence of substrate. After electrophoresis in this “nonbinding mode” the gels are incubated under conditions of optimal enzyme activity to allow substrate hydrolysis to take place before they are stained with substrate-detecting reagents, and active enzymes are detected as clear bands. Multiple forms of a given activity which were resolved during electrophoresis in the nonbinding mode are reflected by the presence of individual bands. The substrate-containing gel electrophoresis technique does not appear to be amenable to precise quantification of enzymes. By comparing the length of the clear tracks or the degree of staining of the activity bands for a range of enzyme concentrations, however, it is possible to establish the smallest amount of enzyme that can unequivocally be detected under a given set of conditions; from such studies we estimate that the sensitivity of detection with the substrate-containing gel electrophoresis technique can be orders of magnitude better than that obtained with other methods. The levels of detection observed in the work presented here were about 50 pg for α-amylase run on starch-containing gels, 1 pg to 1 ng for nucleases run on DNA- or RNA-containing gels, and 100 pg to 10 ng for 11 different pure and crude protease preparations run on gels containing heat-denatured bovine serum albumin.     

De-extinction and the conception of species

Developments in genetic engineering may soon allow biologists to clone organisms from extinct species. The process, dubbed “de-extinction,” has been publicized as a means to bring extinct species back to life. For theorists and philosophers of biology, the process also suggests a thought experiment for the ongoing “species problem”: given a species concept, would a clone be classified in the extinct species? Previous analyses have answered this question in the context of specific de-extinction technologies or particular species concepts. The thought experiment is given more comprehensive treatment here. Given the products of three de-extinction technologies, twenty-two species concepts are “tested” to see which are consistent with the idea that species may be resurrected. The ensuing discussion considers whether or not de-extinction is a conceptually coherent research program and, if so, whether or not its development may contribute to a resolution of the species problem. Ultimately, theorists must face a choice: they may revise their commitments to species concepts (if those concepts are inconsistent with de-extinction) or they may recognize de-extinction as a means to make progress in the species problem.     

On the mechanism of irreversible thermoinactivation of enzymes and possibilities for reactivation of “irreversibly” inactivated enzymes

A possible mechanism for so-called irreversible thermoinactivation of enzymes is suggested. We postulated that in many cases “irreversibly thermoinactivated enzyme” is an incorrectly folded polypeptide chain which, during a reasonable experimental interval, cannot transform into a correctly folded, thermodynamically stable and catalytically active conformation because of high kinetic barriers (intrinsic steric hindrances). Proceeding from this model, reactivation of “irreversibly” thermoinactivated trypsin (covalently attached to CNBr-activated Sepharose to prevent intermolecular processes such as aggregation or autolysis) was carried out. Thermoinactivated trypsin was first transformed to random coil by reduction of its disulphide bonds in 8M urea solution and then renaturated into a catalytically active form by reoxidation of disulphide bonds in the presence of dithiothreitol and in the absence of the denaturant.     

Evolution of regulatory enzymes towards functional simplicity

The potential kinetic complexity of polymeric regulatory enzymes does not seem to be often expressed in nature. Most of these enzymes exhibit in fact a rather simple kinetic behaviour. This functional simplicity is probably the consequence of constraints between rate constants or of blocking of some reaction steps. Functional simplicity is believed to have emerged in the course of neo-Darwinian evolution as a consequence of a trend towards an improved functional efficiency. Functional efficiency may be reached, in polymeric regulatory enzymes, when either of the two sets of conditions are met. The first set of conditions implies the occurrence of the unicity of enzyme conformation in any transition state, a loose coupling between subunits and an exact balance of the driving forces exerted by the enzyme in the forward and backward directions of the catalytic step. This situation results in constraints between rate constants which allow degenerescence of the steady state rate equation. The second set of conditions involves again the unicity of enzyme conformation in any of the transition states, associated with a tight coupling of subunits, and a driving force exerted by the enzyme much strongly in the forward than in the backward direction of the catalytic step. These conditions imply blocking of some reaction steps and again degenerescence of the corresponding rate equation. The most frequent types of quaternary structure and subunit interactions, namely loose coupling between subunits, and tight coupling associated with conservation of at least one symmetry axis, have probably emerged as molecular organizations, which precisely allow both functional efficiency and simplicity to occur. Indeed these situations probably represent the term of two different evolutionary trends. Therefore enzymes that have not reached this state usually exhibit more complex kinetic behaviour. Wavy curves, “bumps” and turning points may be considered as manifestations of the ancestral character of an enzyme.     

Sedimentation of generalized systems of interacting particles. I. Solution of systems of complete Lamm equations.

A very general approach to the chemical equilibria between many interacting molecules during sedimentation (boundary, band, or active enzyme) taking into account boundary conditions, cell geometry, equilibrium constants, diffusion, enzyme kinetics, etc., is presented. Through a Fortran program, the method has been applied to two very simple but typical cases. With only minor adjustments, the method presented here for sedimentation studies can be extended to all sorts of problems in which “pools” of various species are interacting with each other.     

Frontiers in the enzymology of thiamin diphosphate-dependent enzymes

Enzymes that use thiamin diphosphate (ThDP), the biologically active derivative of vitamin B1, as a cofactor play important roles in cellular metabolism in all domains of life. The analysis of ThDP enzymes in the past decades have provided a general framework for our understanding of enzyme catalysis of this protein family. In this review, we will discuss recent advances in the field that include the observation of “unusual” reactions and reaction intermediates that highlight the chemical versatility of the thiamin cofactor. Further topics cover the structural basis of cooperativity of ThDP enzymes, novel insights into the mechanism and structure of selected enzymes, and the discovery of “superassemblies” as reported, for example, acetohydroxy acid synthase. Finally, we summarize recent findings in the structural organisation and mode of action of 2-keto acid dehydrogenase multienzyme complexes and discuss future directions of this exciting research field.     

Competitive coexistence of self-reproducing macromolecules.

Several model systems of self-reproducing molecular species subject to the constraint of constant organization are investigated to see under what conditions more than one species may coexist over extended periods of time. It is found that while coexistence is not possible for systems of simple autocatalytic competitors, it can indeed occur if the nature of the reproductive process gives rise to “catalytic niches”. For systems catalyzed by n Michaelis-Menten type enzymes, it is shown that no more than n species may coexist. A precise characterization is provided of “how different” two such enzymes must be in order to allow coexistence of two species. Under certain conditions, one observes a transition from one dominant species to another which may occur either via a coexistent state or directly, but with hysteresis.     

Ecdysone biosynthesis: Pathways,enzymes, and the early steps problem

Summary

In this review, the different aspects of the ecdysone biosynthesis have been described. The “early step problem” and the enzymes which are involved in the final sequence of hydroxylations are discussed in detail. The recent results obtained on ecdysone biosynthesis in embryos are also summarized.     

Two enzymes in Streptomyces griseus forb the synthesis of dTDP-L-dihydrostreptose from dTDP-6-deoxy-D-xylo-4-hexosulose.

The biosynthesis of dTDP-L-dihydrostreptose from dTDP-6-deoxy-D-$\text{xylo}$-4-hexosulose requires two enzymes: dTDP-4-keto-L-rhamnose-3,5-epimerase and a NADPH-dependent dTDP-“dihydrostreptose synthase”. These enzymes could be separated on a Sephadex G-100 column.     

Induction of the allantoin degradative enzymes by allophanic acid, the last intermediate of the pathway

$\text{Saccharomyces cerevisiae}$ can utilize allantoin as a sole nitrogen source by degrading it in five steps to ammonia, “CO₂”, and glyoxylate. We have previously shown that allophanic acid is the inducer of the urea carboxylase: allophanate hydrolase multienzyme complex. Since these enzymes catalyse the last two steps of allantoin degradation, experiments were performed to determine if allophanate was also the inducer of any other enzymes in the pathway. Our data demonstrate that allophanate induces synthesis of at least five of the seven purine degradative enzymes.     

In vitro maintenance of differentiation marker synthesis by subpopulations of mouse thymocytes

Mouse thymocyte populations enriched in functionally incompetent, “immature” cells on the one hand, or in competent “mature” cells on the other hand, express different steady-state levels of certain surface antigens and marker enzymes. In the cases of the glycoproteins H-2 (K and D), Qa, and TL, and the DNA polymerase terminal deoxynucleotidyl transferase (TdT), these levels reflect different rates of de novo synthesis in the two populations. Thus each population appears to manifest a characteristic pattern of synthetic rates for the various products relative to total protein synthesis. To investigate the maintenance of these patterns, enriched pools of “immature” and “mature” thymocytes were incubated in vitro for 24 h, and the rates of product synthesis before and after culture were compared. H-2 synthesis, initially most rapid in the mature cells, continued to be made at the highest rate in this population. TdT synthesis, a characteristic activity of the immature cells, was not induced in the mature cells, but proceeded at an increased relative rate in the immature population. Therefore, the differences between the rates of H-2 and TdT synthesis were stable properties of the two thymocyte populations. Another marker of immature cells, TL, did not continue to be produced in parallel with TdT. Rather, its synthesis was selectively curtailed in relation to the continuing protein synthesis in the immature cultures. This non-coordinate regulation of TL and TdT production in immature thymocytes may be due to several mechanisms. These are discussed with regard to their implications for pathways of thymocyte maturation.     

A model of pancreatic lipase and the orientation of enzymes at interfaces

A model for the interfacial orientation and the mode of action of lipase is proposed. Lipase is oriented so that its active site is near the oil-water boundary. This orientation is achieved by oil-enzyme bonding at the “hydrophobic head” of the enzyme, a region free of electric charges and relatively resistant to unfolding. The measured K_M is a complex constant including the dissociation constant of this oil-enzyme “complex”. The interfacial orientation of lipase is further aided by hydrophilic negative charges on the “back” of the enzyme and by a hydrophilic carbohydrate “tail”.It is suggested that similar hydrophobic heads and hydrophilic tails and asymmetric charge distributions establish the orientation of many enzymes which act at interfaces. Many phospholipases, for instance, appear to be charge-oriented, and the carbohydrate residues of ribonucleases and many other glycoproteins may be hydrophilic tails.Lipase is probably a serine enzyme with a catalytic center similar to that of chymotrypsin, but more hindered, perhaps owing to the presence of a leucine residue, and there is no binding of substrate lipid chains in the “active complex”. The substrate molecule is fixated on the enzyme in a two-dimensional orientation, because its leaving alkoxy group must be received by the serine hydroxyl hydrogen which is directed towards the imidazol ring of the reactive histidine through a hydrogen bond. The high turnover rate of lipolysis, 5 × 10⁵/min, exceptional even for an enzyme, results from the extremely high substrate concentration near the active site, and from an almost complete extrusion of water because of the hydrophobicity of both the active site and the substrate. In addition, both substrate and enzyme, because of their polarity, are already so favorably positioned at the interface that the formation of the “active complex” requires only a proper two-dimensional alignment, perhaps with partial extraction of the substrate molecule from the lipid phase.     

Regulation of concentrations of glycolytic enzymes and creatine-phosphate kinase in “fast-twitch” and “slow-twitch” skeletal muscles of the chicken

The distinctive contractile and metabolic characteristics of different skeletal muscle fiber types are associated with different protein populations in these cells. In the present work, we investigate the regulation of concentrations of three glycolytic enzymes (aldolase, enolase, glyceraldehyde-3-phosphate dehydrogenase) and creatine-phosphate kinase in “fast-twitch” (breast) and “slow-twitch” (lateral adductor) muscles of the chicken. Results of short-term amino acid incorporation experiments conducted both in vivo and with muscle explants in vitro showed that these enzymes turnover at different rates and that aldolase turns over 2 to 3 times faster than the other three enzymes. However, these differences in turnover rates were difficult to detect in long-term double-isotope incorporation experiments, presumably because extensive reutilization of labeled amino acids occurred during these long-term experiments. Mature muscle fibers synthesize these four cytosolic enzymes at very high rates. For example, 11 to 14% of the total labeled leucine incorporated into protein by breast muscle fibers was found in the enzyme aldolase. Results of short-term amino acid incorporation experiments also showed that the relative rates of synthesis of the three glycolytic enzymes were about fourfold higher in mature “fast-twitch” muscle fibers than in mature “slow-twitch” ones while the relative rates of synthesis of creatine-phosphate kinase were similar in the two fiber types. The relative rates of synthesis of these four enzymes and cytosolic proteins in general were found to be very similar in immature muscles of both types. More profound changes in the relative rates of synthesis of major cytosolic proteins, including the glycolytic enzymes, occurred during postembryonic maturation of fast-twitch fibers than occurred during maturation of slow-twitch fibers. Our work demonstrates that (1) the synthesis of creatine-phosphate is independently regulated with respect to the synthesis of the glycolytic enzymes in muscle fibers; and (2) the approximate fourfold higher steady-state concentrations of glycolytic enzymes in fast-twitch muscle fibers as compared with slow-twitch fibers are determined predominantly by regulatory mechanisms operating at the level of protein synthesis rather than protein degradation. Our demonstration that more profound changes in the relative rates of synthesis of major cytosolic proteins occur during maturation of fast-twitch fibers as compared with slow-twitch fibers is discussed in terms of the mode(s) of fiber-type differentiation proposed by others.     

Ultrastructure of Mucocysts in Peranema trichophorum (Euglenophyceae)1

The “mucigenic” or “muciferous” bodies of Peranema trichophorum are further characterized here as unique extrusive organelles, the mucocysts. Intracellular and ejected mucocysts have characteristic shapes that may represent different developmental stages. Mucocysts found near the Golgi apparatus are membrane-bounded, elongate, tubular structures with amorphous contents of low electron density. Subpellicular mucocysts are often aligned with pellicular striae and have dense contents, which are separated by an electron-lucent zone from granular material at the tips. Ejected mucocysts are uniform in structure and consist of an inner tube with helical striations, an outer tube with a diamond-shaped pattern, and a dense middle band. Fine fibrils, visible only after mucocyst discharge, emanate from the tips. Mucocysts may also protrude through the pellicle and discharge mucilaginous materials into the medium. Acid phosphatase activity is localized within the subpellicular mucocysts, suggesting that they may be involved in release of hydrolytic enzymes into the medium.     

The significance of light activation of enzymes during the induction phase of photosynthesis in isolated chloroplasts

The key reaction of flavonoid biosynthesis, the condensation of the acyl residues from one molecule of 4-coumaroyl-CoA and three molecules of malonyl-CoA, has previously been assumed to be catalyzed by a “flavanone synthase.” Results are presented here which indicate that not the flavanone but the isomeric chalcone is the immediate product of the synthase reaction. The new term “chalcone synthase” is therefore suggested for the enzyme.     

Two-enzyme active transport in vitro with ph induced asymmetrical functional structures.: III. Discussions based on numerical treatments of the time-dependent evolutions

Three complementary models have been considered in which pH gradients (step function. linear pH or linear H^?) impose asymmetry on a two-enzyme mixture. If the “combined pH dependences” of enzymes is pro-asymmetrical, the pH gradient induces an asymmetrical distribution of potential activities (“latent” asymmetry of functional structure). When substrate is added, “developed” asymmetry of effective activities appears which results in “substrate space wave” and pumping when the catalysed reaction couple is “inversible”. It is shown that only one steady state exists for a given boundary condition and is attained when the “combined effective activity” of enzymes is nil: the stationary flux with symmetrical boundaries or the stationary load with moving boundaries is proportional to “effective global activities” of enzymes. “Equivalent square models” could be proposed that would be able to describe “functional” or “permanent” structure pumps as well. These models belong to the thermodynamic branch and the asymmetrical “space wave” substrate concentration profiles obtained must be distinguished from dissipative structures. It appears that such primary active transport pumps are chemical equivalents of heat pumps.     

On optimal nonlinear associative recall

The problem of determining the nonlinear function (“blackbox”) which optimally associates (on given criteria) two sets of data is considered. The data are given as discrete, finite column vectors, forming two matricesX (“input”) andY (“output”) with the same numbers of columns and an arbitrary numbers of rows. An iteration method based on the concept of the generalized inverse of a matrix provides the polynomial mapping of degreek onX by whichY is retrieved in an optimal way in the least squares sense. The results can be applied to a wide class of problems since such polynomial mappings may approximate any continuous real function from the “input” space to the “output” space to any required degree of accuracy. Conditions under which the optimal estimate is linear are given. Linear transformations on the input key-vectors and analogies with the “whitening” approach are also discussed. Conditions of “stationarity” on the processes of whichX andY are assumed to represent a set of sample sequences can be easily introduced. The optimal linear estimate is given by a discrete counterpart of the Wiener-Hopf equation and, if the key-signals are noise-like, the holographic-like scheme of associative memory is obtained, as the optimal nonlinear estimator. The theory can be applied to the system identification problem. It is finally suggested that the results outlined here may be relevant to the construction of models of associative, distributed memory.     

Evolution of enzymes in metabolism: a network perspective

Several models have been proposed to explain the origin and evolution of enzymes in metabolic pathways. Initially, the retro-evolution model proposed that, as enzymes at the end of pathways depleted their substrates in the primordial soup, there was a pressure for earlier enzymes in pathways to be created, using the later ones as initial template, in order to replenish the pools of depleted metabolites. Later, the recruitment model proposed that initial templates from other pathways could be used as long as those enzymes were similar in chemistry or substrate specificity. These two models have dominated recent studies of enzyme evolution. These studies are constrained by either the small scale of the study or the artificial restrictions imposed by pathway definitions. Here, a network approach is used to study enzyme evolution in fully sequenced genomes, thus removing both constraints. We find that homologous pairs of enzymes are roughly twice as likely to have evolved from enzymes that are less than three steps away from each other in the reaction network than pairs of non-homologous enzymes. These results, together with the conservation of the type of chemical reaction catalyzed by evolutionarily related enzymes, suggest that functional blocks of similar chemistry have evolved within metabolic networks. One possible explanation for these observations is that this local evolution phenomenon is likely to cause less global physiological disruptions in metabolism than evolution of enzymes from other enzymes that are distant from them in the metabolic network.