The tricarboxylic acid cycle in Dictyostelium discoideum. I. Formulation of alternative kinetic representations.

Enzyme systems within living cells have recently been shown to be highly ordered structures that violate classic assumptions of the Michaelis-Menten formalism, which originally was developed for the characterization of isolated reactions in vitro. This evidence suggests that a thorough examination of alternative kinetic formalisms for integrated biochemical systems is in order. The purpose of this series of papers is to assess the utility of an alternative power-law formalism by carrying out a detailed comparative analysis of a relatively large, representative system--the tricarboxylic acid cycle of Dictyostelium discoideum. This system was chosen because considerable experimental information already has been synthesized into a detailed kinetic model of the intact system. In this first paper, we set the stage for subsequent analysis within the framework of the power-law formalism: we review the underlying theory, emphasizing recent developments, formulate the model in terms that are convenient for the analysis to follow, and develop the system representation in both the Michaelis-Menten and power-law forms. In the second paper (Shiraishi, F., and Savageau, M. A. (1992) J. Biol. Chem. 267, 22919-22925), these alternative representations are shown to be internally consistent and locally equivalent. The third paper (Shiraishi, F., and Savageau, M. A. (1992) J. Biol. Chem. 267, 22926-22933) provides a complete analysis of the steady state behavior and also treats the dynamic behavior of the model.     

The tricarboxylic acid cycle in Dictyostelium discoideum. III. Analysis of steady state and dynamic behavior.

The examination of model robustness in the previous paper (Shiraishi, F., and Savageau, M. A. (1992) J. Biol. Chem. 267, 22919-22925 led to the suggestion that the current model for the tricarboxylic acid cycle in Dictyostelium discoideum is ill-determined with respect to one or more of the features reflecting pyruvate metabolism. This conclusion is further supported here by results of steady state and dynamic analyses. The tricarboxylic acid cycle, according to the current model, is poised on a knife's edge with its behavior rigidly determined; any alteration of the system's components leads to nonviable behavior, as exemplified by explosive accumulation of pyruvate and loss of steady state in response to a minute change in the level of malate dehydrogenase. With the additional results in this paper, we are able to refine the diagnosis of the problem and suggest three different areas of the current model that might profitably be re-examined by experiment. These include the kinetics of the reactions at the malate branch point, the turnover times for the alanine, glutamate, and aspartate pools in vivo, and the dynamic mass balances for the cofactor NAD. We also suggest a minimal modification in the current model that could alleviate or circumvent some of these problems.     

The tricarboxylic acid cycle in Dictyostelium discoideum. IV. Resolution of discrepancies between alternative methods of analysis.

Experimental studies of enzyme kinetics in vitro and metabolic fluxes in vivo have been used by Wright and her colleagues to develop a detailed kinetic model of the tricarboxylic acid cycle in Dictyostelium discoideum. This model has recently been been analyzed by two different methods (Albe, K. R., and Wright, B. E. (1992) J. Biol. Chem. 267, 3106-3114; Shiraishi, F., and Savageau, M. A. (1992) J. Biol. Chem. 267, 22926-22933 in an effort to determine the response of individual fluxes and metabolite concentrations to changes in levels of the enzymes that constitute the system. Individual responses were found to differ significantly in magnitude as well as in sign. Perhaps the most glaring difference concerns the influence of the enzyme succinate dehydrogenase on the flux through the cycle; in one study, it has the maximum influence, whereas, in the other, it has absolutely no influence. In this paper, we provide a resolution of these discrepancies. We have reconstructed the methodology of Albe and Wright and have been able to reproduce their results in detail. We show that their methodology does not yield a valid steady state analysis, and, consequently, that the conclusions drawn from their analysis must be called into question. First, they concluded that their model is realistic and predictive. It is now clear that their model is ill-determined and has a steady state only for unrealistically narrow conditions. Second, they concluded that their analysis is valid for variations of less than 2% in the levels of the enzymes because they could satisfy summation relationships considered to be mathematically inevitable. It is now clear that these relationships are neither necessary nor sufficient for establishing the validity of an analysis or the appropriateness of a biochemical model. Third, they concluded on the basis of their empirical methodology that certain enzymes are most important in influencing flux through the cycle. It is now clear that these results are inaccurate because of deficiencies in their methodology. Finally, they concluded that steady state analyses cannot be carried out experimentally because of the small variations required in enzyme levels. It is now clear that the requirement for such small variations reflects the ill-determined character of the underlying model and is not a necessary property of the real system.     

Enhanced GTPase activity of transducin when bound to cGMP phosphodiesterase in bovine retinal rods.

The generation of the physiological response of a retinal rod cell to an incident photon involves activation of a cGMP phosphodiesterase (PDE) by a GTP-binding protein, transducin (T). This activation has been shown to occur by formation of a membrane-bound T alpha GTP-PDE complex (Clerc, A., and Bennett, N. (1992) J. Biol. Chem. 267, 6620-6627; Catty, P., Pfister, C., Bruckert, F., and Deterre, P. (1992) J. Biol. Chem 267, 19489-19493). The recovery of the response involves turning-off of T by its intrinsic GTPase activity. We show here that the formation of the membrane-bound T alpha GTP-PDE complex correlates with an enhanced rate of GTP hydrolysis. In vivo, this would provide an appropriate mechanism for fast turn-off of cGMP hydrolysis.     

Lipopolysaccharide induces prostaglandin H synthase-2 in alveolar macrophages.

Prostaglandin H synthase is a key enzyme in the formation of prostaglandins and thromboxane from arachidonic acid. The recent cloning of a second prostaglandin H synthase gene, prostaglandin H synthase-2, which is distinct from the classic prostaglandin H synthase-1 gene, may dramatically alter our concept of how cells regulate prostanoid formation. We have recently shown that the enhanced production of prostanoids by lipopolysaccharide-primed alveolar macrophages involves the induction of a novel prostaglandin H synthase (J. Biol. Chem., (1992), 267, 14547-14550). We report here that the novel PGH synthase induced by lipopolysaccharide in alveolar macrophages is prostaglandin H synthase-2.     

Fluoride-inhibited calcium ATPase of sarcoplasmic reticulum. Magnesium and fluoride stoichiometry.

The sarcoplasmic reticulum (SR) CaATPase is inactivated by fluoride in the presence of magnesium (Murphy, A. J., and Coll, R. J. (1992) J. Biol. Chem. 267, 5229-5235). The inactive complex is very stable and can be isolated free of other components by 48 h of dialysis at 4 degrees C (Murphy, A. J., and Coll, R. J. (1992) J. Biol. Chem. 267, 16990-16994). In this study, we used a fluoride-specific electrode to determine that the amount of tightly bound fluoride in the complex was 9.4 +/- 2 nmol mg-1 SR protein. The rate constant of inactivation was very similar to the rate constant of fluoride incorporation and varied directly as the square of the fluoride concentration. Luminal Ca2+ accelerated reactivation of the inhibited enzyme, and the rate constants of activity regain and fluoride release were very similar. Although required for inhibition, added magnesium did not accelerate reactivation. Analysis for magnesium using antipyrylazo III of the inhibited enzyme showed 4.1 +/- 0.4 nmol mg-1 SR protein. As there is much evidence in the literature supportive of an estimate of calcium pumps equal to approximately 4-5 nmol mg-1 SR protein, our results indicate that each inhibited enzyme contains two tightly bound fluorides and one tightly bound magnesium.     

Proteolytic cleavages of proalbumin and complement Pro-C3 in vitro by a truncated soluble form of furin, a mammalian homologue of the yeast Kex2 protease.

We have recently purified and characterized a truncated soluble form of furin from which the predicted transmembrane domain and cytoplasmic tail were deleted (Hatsuzawa, K., Nagahama, M., Takahashi, S., Takada, K., Murakami, K., and Nakayama, K. (1992) J. Biol. Chem. 267, 16094-16099). Our results showed that furin resembles the yeast Kex2 protease with respect to both its enzymic properties and substrate specificity. Here we demonstrate that the soluble form of furin is capable of converting the precursors of albumin and the third component of complement (proalbumin and pro-C3, respectively) in vitro to mature proteins. Thus furin mimics the Ca(2+)-dependent proalbumin and pro-C3 convertases found in the Golgi membranes (Brennan, S. O., and Peach, R. J. (1988) FEBS Lett. 229, 167-170; Oda, K. (1992) J. Biol. Chem. 267, 17465-17471). Furthermore we show that the variant alpha 1-antitrypsin Pittsburgh, which is a specific inhibitor of the Golgi proalbumin convertase, inhibits not only the Golgi pro-C3 convertase, but also the soluble furin. These results suggest a role for furin in the cleavage of proproteins transported via the constitutive pathway.     

Human trifunctional protein deficiency: a new disorder of mitochondrial fatty acid beta-oxidation.

In this paper we report the identification of a new disorder of mitochondrial fatty acid beta-oxidation in a patient which presented with clear manifestations of a mitochondrial beta-oxidation disorder. Subsequent studies in fibroblasts revealed an impairment in palmitate beta-oxidation and in addition, a combined deficiency of long-chain enoyl-CoA hydratase, long-chain 3-hydroxyacyl-CoA-dehydrogenase and long-chain 3-oxoacyl-CoA thiolase. The recent identification of a multifunctional, membrane-bound beta-oxidation enzyme protein catalyzing all these three enzyme activities (Carpenter et al. (1992) Biochem. Biophys. Res. Commun. 183, 443-448; Uchida et al. (1992) J. Biol. Chem. 267, 1034-1041) suggested an underlying basis for this peculiar combination of three enzyme deficiencies. We show by means of size-exclusion chromatography that there is, indeed, a deficiency of the multifunctional beta-oxidation enzyme protein in this patient.     

The apparent coupling between synthesis and posttranslational modification of Escherichia coli acyl carrier protein is due to inhibition of amino acid biosynthesis.

Acyl carrier protein (ACP) is modified on serine 36 by the covalent posttranslational attachment of 4'-phosphopantetheine from coenzyme A (CoA), and this modification is required for lipid biosynthesis. Jackowski and Rock (J. Biol. Chem 258:15186-15191, 1983) reported that upon depletion of the CoA pool by starvation for a CoA precursor, no accumulation of the unmodified form of ACP (apo-ACP) was detected. We report that this lack of apo-ACP accumulation results from decreased translation of the acpP mRNAs because of the limitation of the synthesis of glutamate and other amino acids made directly from tricarboxylic acid cycle intermediates.     

cDNA cloning of the neutrophil bactericidal peptide indolicidin.

A structurally novel, tryptophan-rich antimicrobial tridecapeptide amide, named indolicidin, has recently been purified from bovine neutrophils (Selsted et al. (1992) J. Biol. Chem. 267, 4292-4295). Here we describe the molecular cloning of this endoantibiotic, which is synthesised in bone marrow cells as a 144 amino acid residue precursor. The encoded protein has a predicted mass of 16479 Da and a pI of 6.51. A putative signal peptide of 29 amino acids precedes a 101 residue pro-region. The mature peptide is at the 3' end of the open reading frame. A glycine, not found in purified indolicidin, is present at the carboxyl terminus of the deduced sequence and is very likely involved in post-translational peptide amidation.     

Evidence that the catalytic activity of prokaryote leader peptidase depends upon the operation of a serine-lysine catalytic dyad.

Leader peptidase (LP) is the enzyme responsible for proteolytic cleavage of the amino acid leader sequence from bacterial preproteins. Recent data indicate that LP may be an unusual serine proteinase which operates without involvement of a histidine residue (M. T. Black, J. G. R. Munn, and A. E. Allsop, Biochem. J. 282:539-543, 1992; M. Sung and R. E. Dalbey, J. Biol. Chem. 267:13154-13159, 1992) and that, therefore, one or more alternative residues must perform the function of a catalytic base. With the aid of sequence alignments, site-specific mutagenesis of the gene encoding LP (lepB) from Escherichia coli has been employed to investigate the mechanism of action of the enzyme. Various mutant forms of plasmid-borne LP were tested for their abilities to complement the temperature-sensitive activity of LP in E. coli IT41. Data are presented which indicate that the only conserved amino acid residue possessing a side chain with the potential to ionize, and therefore with the potential to transfer protons, which cannot be substituted with a neutral side chain is lysine at position 145. The data suggest that the catalytic activity of LP is dependent on the operation of a serine-lysine catalytic dyad.     

Elimination and replenishment of tricarboxylic acid-cycle intermediates in myocardium.

1. The contribution of Co2 fixation to the anaplerotic mechanisms in the myocardium was investigated in isolated perfused rat hearts. 2. K+-induced arrest of the heart was used to elicit a transition in the concentrations of the intermediates of the tricarboxylic acid cycle. 3. Incorporation of 14C from [14]bicarbonate into tricarboxylic acid-cycle intermediates was measured and the rates of the reactions of the cycle were estimated by means of a linear optimization program which solves the differential equations describing a simulation model of the tricarboxylic acid cycle and related reactions. 4. The results showed that the rate of CO2 fixation is dependent on the metabolic state of the myocardium. Upon a sudden diminution of cellular ATP consumption, the pool size of the tricarboxylic acid-cycle metabolites increased and the rate of label incorporation from [14C]bicarbonate into the cycle metabolites increased simultaneously. The computer model was necessary to separate the rapid equilibration between bicarbonate and some metabolites from the potentially anaplerotic reactions. The main route of anaplerosis during metabolite accumulation was through malate + oxaloacetate. Under steady-state conditions there was a constant net outward flow from the tricarboxylic acid cycle via the malate + oxaloacetate pool, with a concomitant anaplerotic flow from metabolites forming succinyl-CoA (3-carboxypropionyl-CoA).     

Identification of two discrete peptide: N-glycanases in Oryzias latipes during embryogenesis.

Two different types of peptide:N-glycanase (PNGase) were identified in developing embryos of medaka fish ( Oryzias latipes ). Because the optimum pH values for their activities were acidic and neutral, they were designated as acid PNGase M and neutral PNGase M, respectively. The acid PNGase M corresponded to the enzyme that had been partially purified from medaka embryos (Seko,A., Kitajima,K., Inoue,Y. and Inoue,S. (1991) J. Biol. Chem., 266, 22110-22114). The apparent molecular weight of this enzyme was 150 K, and the optimal pH was 3.5-4.0, and the K m for L-hyosophorin was 44 microM. L-Hyosophorin is a cortical alveolus-derived glycononapeptide with a large N-linked glycan chain present in the perivitelline space of the developing embryo. Acid PNGase M was competitively inhibited by a free de-N-glycosylated nonapeptide derived from L-hyosophorin. This enzyme was expressed in ovaries and embryos at all developmental stages after gastrulation, but activity was not detected in embryos at developmental stages between fertilization and gastrula. Several independent lines of evidence suggested that acid PNGase M may be responsible for the unusual accumulation of free N-glycans derived from yolk glycoproteins (Iwasaki,M., Seko,A., Kitajima,K., Inoue,Y. and Inoue,S. (1992) J. Biol. Chem., 267, 24287-24296). In contrast, the neutral PNGase M was expressed in blastoderms from the 4-8 cell stage and in cells up to early gastrula. The general significance of these findings is that they show a developmental stage-dependent expression of the two PNGase activities, and that expression of the neutral PNGase M activity occurs concomitantly with the de-N-glycosylation of L-hyosophorin. These data thus support our conclusion that the neutral PNGase M is responsible for the developmental-stage-related de-N-glycosylation of the L-hyosophorin.     

Thermostabilization of Escherichia coli ribonuclease HI by replacing left-handed helical Lys95 with Gly or Asn.

From the systematic replacements of amino acid residues of Escherichia coli ribonuclease HI with those of its thermophilic counterpart, the basic protrusion domain including region 6 (R6) from residues 91 to 95 was found to increase the structural stability of the mutant protein (Kimura, S., Nakamura, H., Hashimoto, T., Oobatake, M., and Kanaya, S. (1992) J. Biol. Chem. 267, 21535-21542). Further mutagenesis concentrating in the R6 region has revealed that replacements of Lys95 at the left-handed structure with Gly or Asn essentially enhances the protein stability. Gly and Asn substitutions stabilize the protein up to 1.9 kcal/mol and 0.9 kcal/mol in the free energy changes of unfolding, respectively. We propose that the amino acid substitution of left-handed non-Gly residue with Gly or Asn residue can be used as one of the general strategies to enhance protein stability, when such a non-Gly residue itself does not seriously contribute to protein stability.     

Mitochondrial energetic metabolism: A simplified model of TCA cycle with ATP production

Mitochondria play a central role in cellular energetic metabolism. The essential parts of this metabolism are the tricarboxylic acid (TCA) cycle, the respiratory chain and the adenosine triphosphate (ATP) synthesis machinery. Here a simplified model of these three metabolic components with a limited set of differential equations is presented. The existence of a steady state is demonstrated and results of numerical simulations are presented. The relevance of a simple model to represent actual in vivo behavior is discussed.     

A new measure of the robustness of biochemical networks

MOTIVATION: The robustness of a biochemical network is defined as the tolerance of variations in kinetic parameters with respect to the maintenance of steady state. Robustness also plays an important role in the fail-safe mechanism in the evolutionary process of biochemical networks. The purposes of this paper are to use the synergism and saturation system (S-system) representation to describe a biochemical network and to develop a robustness measure of a biochemical network subject to variations in kinetic parameters. Since most biochemical networks in nature operate close to the steady state, we consider only the robustness measurement of a biochemical network at the steady state. RESULTS: We show that the upper bound of the tolerated parameter variations is related to the system matrix of a biochemical network at the steady state. Using this upper bound, we can calculate the tolerance (robustness) of a biochemical network without testing many parametric perturbations. We find that a biochemical network with a large tolerance can also better attenuate the effects of variations in rate parameters and environments. Compensatory parameter variations and network redundancy are found to be important mechanisms for the robustness of biochemical networks. Finally, four biochemical networks, such as a cascaded biochemical network, the glycolytic-glycogenolytic pathway in a perfused rat liver, the tricarboxylic acid cycle in Dictyostelium discoideum and the cAMP oscillation network in bacterial chemotaxis, are used to illustrate the usefulness of the proposed robustness measure.     

GalNAc-transferase specificity prediction based on feature selection method

GalNAc-transferase can catalyze the biosynthesis of O-linked oligosaccharides. The specificity of GalNAc-transferase is composed of nine amino acid residues denoted by R4, R3, R2, R1, R0, R1', R2', R3', R4'. To predict whether the reducing monosaccharide will be covalently linked to the central residue R0(Ser or Thr), a new method based on feature selection has been proposed in our work. 277 nonapeptides from reference [Chou KC. A sequence-coupled vector-projection model for predicting the specificity of GalNAc-transferase. Protein Sci 1995;4:1365-83] are chosen for training set. Each nonapeptide is represented by hundreds of amino acid properties collected by Amino Acid Index database (http://www.genome.jp/aaindex) and transformed into a numeric vector with 4554 features. The Maximum Relevance Minimum Redundancy (mRMR) method combining with Incremental Feature Selection (IFS) and Feature Forward Selection (FFS) are then applied for feature selection. Nearest Neighbor Algorithm (NNA) is used to build prediction models. The optimal model contains 54 features and its correct rate tested by Jackknife cross-validation test reaches 91.34%. Final feature analysis indicates that amino acid residues at position R3' play the most important role in the recognition of GalNAc-transferase specificity, which were confirmed by the experiments [Elhammer AP, Poorman RA, Brown E, Maggiora LL, Hoogerheide JG, Kezdy FJ. The specificity of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase as inferred from a database of in vivo substrates and from the in vitro glycosylation of proteins and peptides. J Biol Chem 1993;268:10029-38; O'Connell BC, Hagen FK, Tabak LA. The influence of flanking sequence on the O-glycosylation of threonine in vitro. J Biol Chem 1992;267:25010-8; Yoshida A, Suzuki M, Ikenaga H, Takeuchi M. Discovery of the shortest sequence motif for high level mucin-type O-glycosylation. J Biol Chem 1997;272:16884-8]. Our method can be used as a tool for predicting O-glycosylation sites and for investigating the GalNAc-transferase specificity, which is useful for designing competitive inhibitors of GalNAc-transferase. The predicting software is available upon the request.     

Protein structure of pig liver 4-aminobutyrate aminotransferase and comparison with a cDNA-deduced sequence.

The amino acid sequence of pig liver 4-aminobutyrate aminotransferase has been determined by gas-phase sequencing of proteolytically derived peptide fragments. The sequence differs substantially from that predicted for the same enzyme on the basis of the sequence of cDNA derived from pig brain in recently published work [Kwon, O., Park, J. & Churchich, J. E. (1992) J. Biol. Chem. 267, 7215-7216]. Apart from a few minor differences, the two sequences are completely different in the segment of protein comprising the 36 residues at positions 107-142. Insertion of a cytosine between bases 402 and 403 in the cDNA sequence, together with deletion of the guanine at position 510, results in a DNA sequence which predicts exactly the amino acid sequence determined by peptide analysis in the present work. The mammalian enzyme has approximately 44% sequence identity with the same enzyme from two unicellular eukaryotes (Saccharomyces cerevisiae, Aspergillus nidulans) and 22% identity with that from Escherichia coli.