Spatial order as a source of kinetic cooperativity in organized bound enzyme systems.

When enzyme molecules are distributed within a negatively charged matrix, the kinetics of the conversion of a negatively charged substrate into a product depends on the organization of fixed charges and bound enzyme molecules. Organization is taken to mean the existence of macroscopic heterogeneity in the distribution of fixed charge density, or of bound enzyme density, or of both. The degree of organization is quantitatively expressed by the monovariate moments of charge and enzyme distributions as well as by the bivariate moments of these two distributions. The overall reaction rate of the bound enzyme system may be expressed in terms of the monovariate moments of the charge density and of the bivariate moments of charge and enzyme densities. The monovariate moments of enzyme density do not affect the reaction rate. With respect to the situation where the fixed charges and enzyme molecules are randomly distributed in the matrix, the molecular organization, as expressed by these two types of moments, generates an increase or decrease of the overall reaction rate as well as a cooperativity of the kinetic response of the system. Thus both the alteration of the rate and the modulation of cooperativity are the consequence of a spatial organization of charges with respect to the enzyme molecules. The rate equations have been derived for different types of organization of fixed charges and enzyme molecules, namely, clustered charges and homogeneously distributed enzyme molecules, clustered enzyme molecules and homogeneously distributed charges, clusters of charges and clusters of enzymes that partly overlap, and clusters of enzymes and clusters of charges that are exactly superimposed. Computer simulations of these equations show how spatial molecular organization may modulate the overall reaction rate.     

Mannitol-specific phosphoenolpyruvate-dependent phosphotransferase system of Enterococcus faecalis: molecular cloning and nucleotide sequences of the enzyme IIIMtl gene and the mannitol-1-phosphate dehydrogenase gene, expression in Escherichia coli, and comparison of the gene products with similar enzymes.

Enzyme IIIMtl is part of the mannitol phosphotransferase system of Enterococcus faecalis. It is phosphorylated in a reaction sequence requiring enzyme I and heat-stable phosphocarrier protein (HPr). The phospho group is transferred from enzyme IIIMtl to enzyme IIMtl, which then catalyzes the uptake and concomitant phosphorylation of mannitol. The internalized mannitol-1-phosphate is oxidized to fructose-6-phosphate by mannitol-1-phosphate dehydrogenase. In this report we describe the cloning of the mtlF and mtlD genes, encoding enzyme IIIMtl and mannitol-1-phosphate dehydrogenase of E. faecalis, by a complementation system designed for cloning of gram-positive phosphotransferase system genes. The complete nucleotide sequences of mtlF, mtlD, and flanking regions were determined. From the gene sequences, the primary translation products are deduced to consist of 145 amino acids (enzyme IIIMtl) and 374 amino acids (mannitol-1-phosphate dehydrogenase). Amino acid sequence comparison confirmed a 41% similarity of E. faecalis enzyme IIIMtl to the hydrophilic enzyme IIIMtl-like portion of enzyme IIMtl of Escherichia coli and 45% similarity to enzyme IIIMtl of Staphylococcus carnosus. The putative N-terminal NAD+ binding domain of mannitol-1-phosphate dehydrogenase of E. faecalis shows a high degree of similarity with the N terminus of E. coli mannitol-1-phosphate dehydrogenase (T. Davis, M. Yamada, M. Elgort, and M. H. Saier, Jr., Mol. Microbiol. 2:405-412, 1988) and the N-terminal part of the translation product of S. carnosus mtlD, which was also determined in this study. There is 40% similarity between the dehydrogenases of E. faecalis and E. coli over the whole length of the enzymes. The organization of mannitol-specific genes in E. faecalis seems to be similar to the organization in S. carnosus. The open reading frame for enzyme IIIMtl E. faecalis is followed by a stem-loop structure, analogous to a typical Rho-independent terminator. We conclude that the mannitol-specific genes are organized in an operon and that the gene order is mtlA orfX mtlF mtlD.     

Molecular organization and clustering of cell-wall-bound enzymes as a source of kinetic apparent co-operativity

When fixed charges and enzyme molecules are not homogeneously distributed in a matrix, the degree of organization of charges, of enzyme molecules and of charges with respect to enzyme molecules modulate the enzyme reaction rate. The overall reaction velocity of the bound enzyme system may be expressed in terms of monovariate moments of the charge density distribution and of the bivariate moments of the charge and enzyme density distributions. With respect to the situation where fixed charges and enzyme molecules are randomly distributed in the matrix, the molecular organization, as expressed by the monovariate and bivariate moments results in an increase or a decrease, of the overall reaction rate, as well as in the appearance of a kinetic cooperativity. The degree of spatial organization of objects may be expressed quantitatively through the concept of minimal spanning tree. This concept may thus be applied to the quantification of the degree of order that may exist in the bidimensional distribution of enzyme molecules in a charged matrix. Primary walls of isolated plant cells in sterile culture behave as a polyanion and contain different enzymes. The spatial distribution in sycamore cell walls of an acid phosphatase has been studied through the concept of minimal spanning tree and shown to be non-randomly distributed in the polyanionic matrix, but clustered in that matrix. This spatial organization results in a modulation of the reaction rate of the cell-wall-bound phosphatase reaction. Both the theoretical and experimental results presented in this study leave little doubt as to the validity of the idea that in situ the organization of fixed charges and enzyme molecules modulate the overall dynamics of enzyme reactions.     

Sequence organization of the nuclear DNA of Schizophyllum commune

Several methods were used to characterize the organization of repetitive DNA in the fungus Schizophyllum commune. They all failed to show interspersion of repetitive sequences among single copy sequences. Saturation hybridization showed that 2.2% of the double-stranded nuclear DNA coded for rRNA. The size of the ribosomal cistron (11.9.10(6) daltons) was determined by restriction enzyme analysis. From these values it was calculated that about 6% of the nuclear DNA consisted of ribosomal cistrons, which approx. equals the amount of repetitive DNA present. Thus, this simple sequence organization in Schizophyllum commune is fundamentally different from organization patterns in higher eukaryotes.     

Targeted protein accumulation promoted by autoassembly and its recovery from plant cells

The expression of a fusion protein formed between the avian infectious bronchitis virus M protein and the bacterial enzyme beta-glucuronidase (GUS) in plants promotes the formation of new organization of the endoplasmic reticulum in tobacco plants. This unusual organization of the membranes, never present in nontransformed plants, has been explained by the oligomerization of the GUS domains of the IBVM-GUS fusion proteins. These specific organized membranes could have broad implications for biotechnology since their formation could be used as a mechanism for retaining and accumulating resident proteins in specific and discrete membrane compartments. In this study, we have shown that the unusual organization of native membranes due to overexpression of the IBVM-GUS fusion gene in tobacco transgenic plants and calli is present at higher levels in plant cell suspensions than in plant tissues. In these cell suspensions, IBVM-GUS protein was continuously synthesized and accumulated throughout the cell culture. An enrichment of the chimeric IBVM-GUS protein corresponding to a five-fold increase in the microsomal fractions was achieved and the GUS enzyme did not show any modification on enzyme kinetics. However, the GUS activity could be differentially distributed in the fractions eluted at different pH suggesting differences in the surface topography of histidine residues for this recombinant GUS.     

Engineering the spatial organization of metabolic enzymes: mimicking nature's synergy

A growing body of evidence indicates that many cellular reactions within metabolic pathways are catalyzed not by free-floating 'soluble' enzymes, but via one or more membrane-associated multienzyme complexes. This type of macromolecular organization has important implications for the overall efficiency, specificity, and regulation of metabolic pathways. An ever-increasing number of biochemical and genetic studies on primary and secondary metabolism have laid a solid foundation for this model, providing compelling evidence in favor of the so-called channeling of intermediates between enzyme active sites and colocalization of enzymes inside a cell. In this review, we discuss several of nature's most notable multifunctional enzyme systems including the AROM complex and tryptophan synthase, each of which provides new fundamental insights into the structural organization of metabolic machinery within living cells. We then focus on the growing body of literature related to engineering strategies using protein chimeras and post-translational assembly mechanisms. Common among these techniques is the desire to mimic natural enzyme organization for optimizing the production of valuable metabolites with industrial and medical importance.     

Transient-time analysis of substrate-channelling in interacting enzyme systems.

The kinetics of dynamically interacting enzyme systems is examined, in the light of increasing evidence attesting to the widespread occurrence of this mode of organization in vivo. The transient time, a key phenomenological parameter for the coupled reaction, is expressed as a function of the lifetime of the intermediate substrate. The relationships between the transient time and the pseudo-first-order rate constants for the coupled reaction by the complexed and uncomplexed enzyme species are indicative of the mechanism of intermediate transfer ('channelling'). In a dynamically interacting enzyme system these kinetic parameters are composite functions of those for the processes catalysed by the complex and by the separated enzymes. The mathematical paradigm can be extended to a linear sequence of N coupled reactions catalysed by dynamically (pair-wise) interacting enzymes.     

The liver/erythrocyte pyruvate kinase gene complex [Pk-1] in the mouse: regulatory gene mutations.

Nine enzyme activity variants and one charge variant of liver/erythrocyte pyruvate kinase have been found amongst laboratory and wild mice. Four of the enzyme activity variants were previously reported to be caused by allelic differences in the structural gene, Pk-1s. Analysis of two putative regulatory gene mutations is now reported, both of which map at, or close to, the structural gene on chromosome 3. One of these mutations, in the inbred strain SWR, is tissue specific, affecting enzyme concentration in the liver but not the erythrocyte the other, which arose in a mutation experiment, doubles the enzyme concentration in both tissues. The organization and the nomenclature in the [Pk-1] gene complex are discussed and are compared with the organization of other comprehensively analysed gene complexes in the mouse.     

Purification of Phosphomannanase and Its Action on the Yeast Cell Wall

An improved assay for phosphomannanase (an enzyme required for the preparation of yeast protoplasts) has been developed based on the release of mannan from yeast cell walls. A procedure for the growth of Bacillus circulans on a large scale for maximal production of the enzyme is described. The culture medium containing the secreted enzyme was concentrated, and the enzyme was purified by protamine sulfate treatment, ammonium sulfate fractionation, gel filtration on P-100, and isoelectric density gradient electrophoresis. Although the enzyme was purified to apparent homogeneity, it still contained laminarinase activity which could not be separated by size or charge. The two enzymatic activities also exhibited two isoelectric points (pH 5.9 and 6.8) on ampholine electrophoresis. The laminarinase was not active on yeast glucan. The enzyme preparation was shown to remove mannan from yeast without removing glucan. Electron microscopic observation supports the idea that this mannan is the outer layer of the yeast wall. Phosphomannanase will produce protoplasts from yeast when supplemented with relatively low amounts of snail enzyme. This activity is present in snail enzyme but appeares to be rate-limiting when snail enzyme alone is used. Phosphomannanase has proven useful for studying the macromolecular organization of polymers in the yeast cell wall.     

Purification and characterization of a highly unusual tetrameric D-lactate dehydrogenase from the muscle of the giant barnacle, Balanus nubilus Darwin

d-Lactate dehydrogenase from the depressor muscle of the giant barnacle, Balanus nubilus Darwin, was purified to homogeneity. The molecular weight of this enzyme, as judged by meniscus depletion sedimentation equilibrium and gel filtration, corresponds to a tetrameric subunit organization unlike the d-lactate dehydrogenases from the horeseshoe crab, Limulus polyphemus, and the polychaete, Nereis virens, which are dimeric. It is concluded that substrate stereospecificity and the degree of subunit organization are two independent parameters in the evolution of lactate dehydrogenases. The amino acid composition of B. nubilusd-lactate dehydrogenase shows general similarities to both the Limulus enzyme and the l-lactate dehydrogenase from the lobster, Homarus americanus, except for an unusually high cysteine content (10 residues per subunit). The isoelectric point of the barnacle enzyme is 5.0. B. nubilusd-lactate dehydrogenase is clearly a muscle-type enzyme, as it displays very little substrate inhibition at high pyruvate concentrations. The catalytic properties of this enzyme, including high reactivity with α-ketobutyrate and α-hydroxybutyrate, lowered pH optimum (7.5) for lactate oxidation, and relative insensitivity to oxamate, also set it apart from other animal d-lactate dehydrogenases.     

EPR studies of iron-sulfur clusters in isolated subunits and subfractions of NADH-ubiquinone oxidoreductase

The thermodynamic and EPR characteristics of the iron-sulfur clusters of NADH-ubiquinone oxidoreductase have been examined in various subfractions and subunits of the enzyme. These were obtained by fragmentation of the enzyme with chaotropic agents and detergent and salt fractionation. We provide evidence for the presence of three tetranuclear clusters and five or six binuclear clusters, accounting well for the chemically determined iron content of this enzyme (22-24 atoms/molecule of FMN). Some of the clusters can be identified with EPR-detectable species in intact NADH-ubiquinone oxidoreductase and, by combining information on subunit topography and spin-spin interactions between redox centers in the native enzyme, we propose a tentative scheme for the spatial organization of these iron-sulfur clusters in the enzyme and in the membrane.     

Conformational change of rabbit aminopeptidase N into enterocyte plasma membrane domains analyzed by flow cytometry fluorescence energy transfer

Membrane vesicle preparations are very appropriate material for studying the topology of glycoproteins integrated into specialized plasma membrane domains of polarized cells. Here we show that the flow cytometric measurement of fluorescence energy transfer used previously to study the relationship between surface components of isolated cells can be applied to membrane vesicles. The fluorescein and rhodamine derivatives of a monoclonal antibody (4H7.1) that recognized one common epitope of the rabbit and pig aminopeptidase N were used for probing the oligomerization and conformational states of the enzyme integrated into the brush border and basolateral membrane vesicles prepared from rabbit and pig enterocytes. The high fluorescent energy transfer observed in the case of pig enzyme integrated into both types of vesicles and in the case of the rabbit enzyme integrated into basolateral membrane vesicles agreed very well with the existence of a dimeric organization, which was directly demonstrated by cross-linking experiments. Although with the latter technique we observed that the rabbit aminopeptidase was also dimerized in the brush border membrane, no energy transfer was detected with the corresponding vesicles. This indicates that the relative positions of two associated monomers differ depending on whether the rabbit aminopeptidase is transiently integrated into the basolateral membrane or permanently integrated into the brush border membrane. Cross-linking of aminopeptidases solubilized by detergent and of their ectodomains liberated by trypsin showed that only interactions between anchor domains maintained the dimeric structure of rabbit enzyme whereas interactions between ectodomains also exist in the pig enzyme. This might explain why the noticeable change in the organization of the two ectodomains observed in the case of rabbit aminopeptidase N does not occur in the case of pig enzyme.     

Markov chain methods in enzyme kinetics

An enzyme cluster is a system of enzymes attached to a membrane, whose spatial organization makes it possible for the product of one enzymatic reaction tobe directly available as a substrate of another reaction within the cluster. We show how to model enzyme clusters by Markov chains, and how to compute their overall reaction rate. As a by-product we prove a formula for the number of completed cycles per unit time in a Markov chain.     

The mitochondrial adenosine triphosphatase of Acanthamoeba castellanii. Oscillatory accumulation of enzyme activity, enzyme protein and F1-inhibitor during the cell cycle.

1. The mitochondrial ATPase of Acanthamoeba castellanii accumulated discontinuously in synchronous cultures prepared by a minimally perturbing size-selection technique. 2. Enzyme activity per ml of culture doubled overall during one cell cycle time of 8 h, but oscillated to give seven maxima during this period. Similar oscillations were observed in the specific activities of ATPase and of the naturally occurring inhibitor protein. 3. These variations in enzyme activity reflected changes in amount of enzyme protein as assayed by an immunological technique. 4. Large variations in I50 values (micrograms of inhibitor/mg of protein necessary for 50% inhibition of inhibitor-sensitive activity) for inhibition of ATPase activity by seven different inhibitors of energy conservation were observed. Activity was more sensitive to inhibition by oligomycin, efrapeptin, citreoviridin and quercetin when values were highest. 5. The results are discussed in relation to the phased organization of biosynthesis and degradation of cellular components known to occur during the cell cycle of this organization.     

The crystal structure of human GlnRS provides basis for the development of neurological disorders

Cytosolic glutaminyl-tRNA synthetase (GlnRS) is the singular enzyme responsible for translation of glutamine codons. Compound heterozygous mutations in GlnRS cause severe brain disorders by a poorly understood mechanism. Herein, we present crystal structures of the wild type and two pathological mutants of human GlnRS, which reveal, for the first time, the domain organization of the intact enzyme and the structure of the functionally important N-terminal domain (NTD). Pathological mutations mapping in the NTD alter the domain structure, and decrease catalytic activity and stability of GlnRS, whereas missense mutations in the catalytic domain induce misfolding of the enzyme. Our results suggest that the reduced catalytic efficiency and a propensity of GlnRS mutants to misfold trigger the disease development. This report broadens the spectrum of brain pathologies elicited by protein misfolding and provides a paradigm for understanding the role of mutations in aminoacyl-tRNA synthetases in neurological diseases.     

The carbamoyl-phosphate synthetase of Pyrococcus furiosus is enzymologically and structurally a carbamate kinase.

The hyperthermophiles Pyrococcus furiosus and Pyrococcus abyssi make pyrimidines and arginine from carbamoyl phosphate (CP) synthesized by an enzyme that differs from other carbamoyl-phosphate synthetases and that resembles carbamate kinase (CK) in polypeptide mass, amino acid sequence, and oligomeric organization. This enzyme was reported to use ammonia, bicarbonate, and two ATP molecules as carbamoyl-phosphate synthetases to make CP and to exhibit bicarbonatedependent ATPase activity. We have reexamined these findings using the enzyme of P. furiosus expressed in Escherichia coli from the corresponding gene cloned in a plasmid. We show that the enzyme uses chemically made carbamate rather than ammonia and bicarbonate and catalyzes a reaction with the stoichiometry and equilibrium that are typical for CK. Furthermore, the enzyme catalyzes actively full reversion of the CK reaction and exhibits little bicarbonate-dependent ATPase. In addition, it cross-reacts with antibodies raised against CK from Enterococcus faecium, and its three-dimensional structure, judged by x-ray crystallography of enzyme crystals, is very similar to that of CK. Thus, the enzyme is, in all respects other than its function in vivo, a CK. Because in other organisms the function of CK is to make ATP from ADP and CP derived from arginine catabolism, this is the first example of using CK for making rather than using CP. The reasons for this use and the adaptation of the enzyme to this new function are discussed.     

Cortical microtubule organization in Vitis protoplasts as affected by concentration of enzyme isolation medium and duration of incubation

Protoplasts of Vitis rotundifolia Michx. cv. Summit were isolated from mesophyll of axenic shoot cultures under different enzyme concentrations and digestion times. Viability and plating efficiency were assessed and related to the cortical microtubule network, visualized using immunofluorescence. Higher concentrations of enzyme isolation medium significantly decreased protoplast viability and plating efficiency. However, the cortical microtubule network appeared stable, at all concentrations with dense, continuous microtubule strands in both random and parallel arrays. In contrast, longer vs shorter enzyme incubation duration resulted in significantly lower plating efficiency, which was correlated with changes in cortical microtubule organization. With longer incubation, the frequency of parallel microtubule strands decreased; microtubule organization showed increasing disruption, microtubule strands were shortened, fragmented and exhibited only a weak fluorescence labeling. Both high enzyme concentration and prolonged incubation periods negatively affected protoplast regenerability, but in different ways. Microtubule organization was sensitive to duration of incubation, but not to enzyme concentration. It is concluded that the presence of a well-developed cortical microtubule network does not gurantee regeneration. Other factors related to isolation appear to be involved.     

Thyroxine-dependent modulation of actin polymerization in cultured astrocytes. A novel, extranuclear action of thyroid hormone

Actin depolymerization specifically blocks the rapid thyroid hormone-dependent inactivation of type II iodothyronine 5'-deiodinase. Thyroid hormone appears to regulate enzyme inactivation by modulating actin-mediated internalization of this plasma membrane-bound protein. In this study, we examined the interrelationships between thyroxine-dependent enzyme inactivation and the organization of the actin cytoskeleton in cultured astrocytes. Steady-state enzyme levels were inversely related to actin content in dibutyryl cAMP-stimulated astrocytes, and increases in filamentous actin resulted in progressively shorter enzyme half-lives without affecting enzyme synthesis. In the absence of thyroxine, filamentous actin decreased by approximately 40% and soluble actin correspondingly increased; thyroxine normalized filamentous actin levels without changing total cell actin. Thyroxine treatment for only 10 min resulted in an approximately 50% loss of enzyme and increased filamentous actin 2-fold. Neither cycloheximide nor actinomycin D affected the thyroxine-induced actin polymerization. Astrocytes grown without thyroxine also showed a disorganized actin cytoskeleton, and 10 nM thyroxine or 10 nM reverse triiodothyronine normalized the actin cytoskeleton appearance within 20 min; 10 nM 3,3',5-triiodothyronine had no effect. These data show that thyroxine modulates the organization of the actin cytoskeleton in astrocytes and suggest that regulation of actin polymerization may contribute to thyroid hormone's influence on arborization, axonal transport, and cell-cell contact in the developing brain.