Organization of enzymes on subcellular structures: relay at the surface

There is a large body of evidence that soluble cytoplasmic enzymes of eukaryotic cells, e.g., glycolytic enzymes and proteins of the translational machinery, are organized in some way in space and in time. The following features of such organization emerge from the experimental data: (1) metabolites are transferred between enzymes directly "from hand to hand" in short-living enzyme-enzyme complexes rather than by diffusion in aqueous media; (2) enzymes show a tendency to be absorbed on surfaces of subcellular structures, such as membranes, cytoskeleton and polyribosomes; (3) enzymes are desorbed from a surface of a subcellular structure after binding specific metabolites, i.e., substrates and/or products of the reactions catalyzed by these enzymes. These features are suggestive of a relay mechanism for the enzyme systems functioning in a cell; an enzyme adsorbed on a surface of a subcellular structure is desorbed after binding its substrate or in the course of the catalytic act. Within a complex with its product the enzyme diffuses into the environment, until it reaches the next enzyme adsorbed on the same surface; then a short-living enzyme-enzyme complex is formed, and a direct "from hand to hand" transfer of the metabolite takes place. As a result, the overall metabolic process appears to be localized near the surface. We termed this mechanism as a "relay at the surface".     

Where is the glycolytic complex? A critical evaluation of present data from muscle tissue

Associations between glycolytic enzymes and subcellular structures have been interpreted as presenting a novel mechanism of glycolytic control; reversible enzyme binding to subcellular structural components is believed to regulate enzyme activity in vivo through the formation of a multi-enzyme complex. However, three lines of evidence suggest that enzyme binding to cellular structures is not involved in the control of glycolysis. (i) Calculations of the distribution of glycolytic enzymes under the physiological cellular conditions of higher ionic strength and higher enzyme concentrations indicate that a large multi-enzyme complex would not exist. (ii) In many cases, binding to subcellular structures is accompanied by changes in enzyme kinetic parameters brought about by allosteric modification, but these changes often inhibit enzyme activity. (iii) In the case where formation of binary enzyme/enzyme complexes activates enzymes, the overall increase in flux through the enzyme reaction is negligible.     

Aminopropyltransferases Involved in Polyamine Biosynthesis Localize Preferentially in the Nucleus of Plant Cells

Plant aminopropyltransferases consist of a group of enzymes that transfer aminopropyl groups derived from decarboxylated S-adenosyl-methionine (dcAdoMet or dcSAM) to propylamine acceptors to produce polyamines, ubiquitous metabolites with positive charge at physiological pH. Spermidine synthase (SPDS) uses putrescine as amino acceptor to form spermidine, whereas spermine synthase (SPMS) and thermospermine synthase (TSPMS) use spermidine as acceptor to synthesize the isomers spermine and thermospermine respectively. In previous work it was shown that both SPDS1 and SPDS2 can physically interact with SPMS although no data concerning the subcellular localization was reported. Here we study the subcellular localization of these enzymes and their protein dimer complexes with gateway-based Bimolecular Fluorescence Complementation (BiFC) binary vectors. In addition, we have characterized the molecular weight of the enzyme complexes by gel filtration chromatography with in vitro assembled recombinant enzymes and with endogenous plant protein extracts. Our data suggest that aminopropyltransferases display a dual subcellular localization both in the cytosol and nuclear enriched fractions, and they assemble preferably as dimers. The BiFC transient expression data suggest that aminopropyltransferase heterodimer complexes take place preferentially inside the nucleus.     

Degradation of Thyrotropin-Releasing Hormone and Luteinising Hormone-Releasing Hormone by Enzymes of Brain Tissue

Abstract: In this article, the enzymes of brain and associated tissues that can degrade thyrotropin-releasing hormone (TRH) and luteinising hormone-releasing hormone (LH-RH) are reviewed. As both TRH and LH-RH are considered to act as neurotransmitters or neuromodulators in the CNS, attention is paid to the subcellular location of the enzymes described and how their topographies and substrate specificities fit them to playing roles as inactivating agents for TRH and LH-RH or as regulators of intracellular concentrations of TRH and LH-RH. Consideration is also given to enzymes involved in biotransformation of TRH to secondary metabolites that exhibit biological activity and to enzymes involved in the metabolism of secondary metabolites.     

Studies on the nature of the subcellular localization of lactate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase in chicken skeletal muscle

The elucidation of the subcellular localization of enzymes by the classical technique of homogenization followed by differential centrifugation is limited in that it is difficult to determine the effect of the severe disruptive procedures on the normal relationship of the enzymes to their subcellular environment. Attempts have been made to study this problem under less severe limitations; one of the approaches used has been the use of pressure on whole muscle tissue to extract the cellular fluids. In this report we introduce the concept of “comparative extraction” for evaluation of results obtained by this procedure. By comparing the efflux of enzymes of similar solubility and similar size and shape, it is possible to determine the minimal amount of the less easily extractable enzyme which cannot be removed due to compartmentation or binding to cellular particulate structures. Using this concept of “comparative extraction,” we show in this report that at least 35% of the lactate dehydrogenase of chicken breast muscle is restricted in its removal. The data do not definitely resolve the problem of whether the restriction is due to compartmentation of the enzyme within subcellular organelles or binding to subcellular structures.     

Analysis of the compartmentation of glycolytic intermediates, nucleotides, sugars, organic acids, amino acids, and sugar alcohols in potato tubers using a nonaqueous fractionation method

The compartmentation of metabolism in heterotrophic plant tissues is poorly understood due to the lack of data on metabolite distributions and fluxes between subcellular organelles. The main reason for this is the lack of suitable experimental methods with which intracellular metabolism can be measured. Here, we describe a nonaqueous fractionation method that allows the subcellular distributions of metabolites in developing potato (Solanum tuberosum L. cv Desiree) tubers to be calculated. In addition, we have coupled this fractionation method to a recently described gas chromatography-mass spectrometry procedure that allows the measurement of a wide range of small metabolites. To calculate the subcellular metabolite concentrations, we have analyzed organelle volumes in growing potato tubers using electron microscopy. The relative volume distributions in tubers are very similar to the ones for source leaves. More than 60% of most sugars, sugar alcohols, organic acids, and amino acids were found in the vacuole, although the concentrations of these metabolites is often higher in the cytosol. Significant amounts of the substrates for starch biosynthesis, hexose phosphates, and ATP were found in the plastid. However, pyrophosphate was located almost exclusively in the cytosol. Calculation of the mass action ratios of sucrose synthase, UDP-glucose pyrophosphorylase, phosphoglucosisomerase, and phosphoglucomutase indicate that these enzymes are close to equilibrium in developing potato tubers. However, due to the low plastidic pyrophosphate concentration, the reaction catalyzed by ADP-glucose pyrophosphorylase was estimated to be far removed from equilibrium.     

Metabolites of benzo(a) pyrene produced by placental microsomes from cigarette smokers and nonsmokers

Placental microsomes from a large majority of nonsmokers in this study showed almost undetectable specific activity in metabolizing benzo(a)pyrene (BaP) in vitro. The microsomal fraction of the placentas from individuals who smoked cigarettes during pregnancy had the highest activity in metabolizing BaP than other subcellular fractions. Cigarette smoking during pregnancy induced placental enzymes which converted BaP to a variety of metabolites: the yield of 3-hydroxy-BaP (3-OHBaP) and other phenols of BaP was the largest among the BaP metabolites, 7,8-dihydrodihydroxy-BaP (7,8-diol) having 13–72% the yield of 3-OHBaP. Other metabolites included 9, 10-dihydrodihydroxy-BaP (9,10-diol), 4,5-dihydrodihydroxy-BaP (4,5-diol), quinones of BaP and unidentified metabolites which were more polar than the diols.     

Subcellular fractionation of isolated rat hepatocytes a comparison with liver homogenate

An improved method for the homogenization and the subsequent subcellular fractionation of hepatocytes isolated from adult rat liver is described.The homogenization procedure developed in the present study allows the preservation of the integrity of subcellular structures, as demonstrated by measurement of the activities of representative enzymes as well as by determination of their latency.The activities of representative marker enzymes, as calculated on subcellular fractions obtained by differential centrifugation of the homogenate, are identical whether the homogenate arises from isolated hepatocytes or from the whole liver.Moreover, there is a close similitude between the kinetic parameters (K_{m and} V) of two microsomal cytochrome P₄₅₀-dependent mixed-function oxidases, namely aniline hydroxylase and aminopyrine demethylase determined on microsomal preparations obtained either from isolated cells or from the whole liver.     

A rapid method for the preparation of microvilli from rabbit kidney

A simple method for the isolation of microvilli from kidney brush border is described. The method depends on the preferential aggregation of other subcellular structures by bivalent metal ions. MgCl(2) is added to a homogenate of cortical tissue prepared from frozen rabbit kidneys. Aggregated material is removed by a low-speed centrifugation and the supernatant centrifuged at 15000g to yield a pellet enriched in microvilli. This is resuspended and given a second treatment with Mg(2+). The purified preparation is obtained after four short differential centrifugations. The six brush-border enzymes that were monitored were enriched 11-17-fold compared with the original homogenate and were obtained in about 10% yield. Marker enzymes for other subcellular components showed the preparation to be essentially free of mitochondria and to be less contaminated with endoplasmic reticulum and baso-lateral plasma membranes than are conventional brush-border preparations. The main contamination was of lysosomal origin, about half of which was attributable to adsorbed acid hydrolases rather than to intact lysosomes. The aggregated components in the low-speed pellet bound less Mg(2+) than did the microvillus fraction. A possible mechanism for the role of Mg(2+) is discussed.     

Mathematical modelling of the urea cycle. A numerical investigation into substrate channelling.

Metabolite channelling, the process in which consecutive enzymes have confined substrate transfer in metabolic pathways, has been proposed as a biochemical mechanism that has evolved because it enhances catalytic rates and protects unstable intermediates. Results from experiments on the synthesis of radioactive urea [Cheung, C., Cohen, N.S. & Raijman, L (1989) J. Biol. Chem.264, 4038-4044] have been interpreted as implying channelling of arginine between argininosuccinate lyase and arginase in permeabilized hepatocytes. To investigate this interpretation further, a mathematical model of the urea cycle was written, using Mathematica it simulates time courses of the reactions. The model includes all relevant intermediates, peripheral metabolites, and subcellular compartmentalization. Analysis of the output from the simulations supports the argument for a high degree of, but not absolute, channelling and offers insights for future experiments that could shed more light on the quantitative aspects of this phenomenon in the urea cycle and other pathways.     

Cytotoxicity and effect of glycyl-D-phenylalanine-2-naphthylamide on lysosomes

Glycyl-D-phenylalanine-2-naphthylamide (Gly-D-Phe-2-NNap) is a cytotoxic agent as exemplified by its effect on Vero cells in culture. This effect is inhibited to some extent by nigericin. On the other hand, Gly-D-Phe-2-NNap induces an increase of free activity of N-acetylglucosaminidase when incubated with a mitochondrial fraction of rat liver at pH 7.5. The phenomenon is inhibited by chloroquine, NH4Cl and nigericin, substances that are known to increase the intralysosomal pH. The latency of enzymes located in other subcellular structures - mitochondria, peroxisomes and endoplasmic reticulum - is not affected by Gly-D-Phe-2-NNap. Moreover, that compound does not cause a release of FITC-Dextran present in endosomes. Apparently Gly-D-Phe-2-NNap is a specific lytic agent for lysosomes. It is proposed that the molecule behaves like a lysosomotropic substance that is able to attack the lysosomal membrane from the interior of the organelle. Its cytotoxic properties could be explained by its effect on lysosomes.     

Adsorption of peripheral enzymes to membrane anchor proteins

The character of the isotherms of specific adsorption of peripheral enzymes to dimeric anchor proteins embedded in the membrane has been analysed. The situations are discussed when adsorption corresponds to the stoichiometry of one or two molecules of peripheral enzyme per dimeric binding site. The corresponding expressions describing the competitive interrelationships between peripheral enzymes adsorbed to the same binding sites have been derived. The experimental data on the adsorption of glycolytic enzymes to erythrocyte membranes are used for the illustration of the theoretical predictions. The physiological role of enzyme self-association which leads to the formation of enzyme oligomers of unlimited length is discussed. It is assumed that under in vivo conditions the association sites of such enzymes are saturated through interactions with anchor proteins of subcellular structures and with the enzymes of the corresponding metabolic pathways. Therefore the linearly associating enzymes play the key role in the formation of multienzyme complexes attached to subcellular structures. The significance of 6-phosphofructokinase adsorption to erythrocyte membranes in the formation of the complex of glycolytic enzymes is discussed.     

Glycosyltransferases: managers of small molecules

Studies of the glycosyltransferases (GTs) of small molecules have greatly increased in recent years as new approaches have been used to identify their genes and characterize their catalytic activities. These enzymes recognize diverse acceptors, including plant metabolites, phytotoxins and xenobiotics. Glycosylation alters the hydrophilicity of the acceptors, their stability and chemical properties, their subcellular localisation and often their bioactivity. Considerable progress has been made in understanding the role of GTs in the plant and the utility of GTs as biocatalysts, the latter arising from their regio- and enantioselectivity and their ability to recognize substrates that are not limited to plant metabolites.     

A cut above the rest: the regulatory function of plant proteases

Proteolytic enzymes are intricately involved in many aspects of plant physiology and development. On the one hand, they are necessary for protein turnover. Degradation of damaged, misfolded and potentially harmful proteins provides free amino acids required for the synthesis of new proteins. Furthermore, the selective breakdown of regulatory proteins by the ubiquitin/proteasome pathway controls key aspects of plant growth, development, and defense. Proteases are, on the other hand, also responsible for the post-translational modification of proteins by limited proteolysis at highly specific sites. Limited proteolysis results in the maturation of enzymes, is necessary for protein assembly and subcellular targeting, and controls the activity of enzymes, regulatory proteins and peptides. Proteases are thus involved in all aspects of the plant life cycle ranging from the mobilization of storage proteins during seed germination to the initiation of cell death and senescence programs. This article reviews recent findings for the major catalytic classes, i.e. the serine, cysteine, aspartic, and metalloproteases, emphasizing the regulatory function of representative enzymes.     

The association of glycolytic enzymes with cellular and model membranes

This article deals with the binding of glycolytic enzymes with membranous or protein subcellular structures. The representative papers of the last three decades dealing with this matter are reviewed. The studies evidencing the binding of some glycolytic enzymes to insoluble subcellular proteins and membranous structures are presented. It is currently generally accepted that the glycolytic enzymes work in some organisation. Such organisation undoubtedly plays a marked role, although still poorly known, in the regulation processes of glycolysis. From this review, the conclusion emerges that the regulatory ability of the binding of glycolytic enzymes to cellular membranes should be added to the list of well-known mechanisms of post-translational regulation of the glycolytic enzymes. Some of the results presented are the background for the hypothesis that planar phospholipid domains in/on the membrane surface are capable of functioning as binding sites for these enzymes. Such binding can modify the conformation state of the enzymes, which results in changes in their kinetic properties; thus, it may function as a regulator of catalytic activity     

In vivo analysis of metabolic dynamics in Saccharomyces cerevisiae: II. Mathematical model

A mathematical model of glycolysis in Saccharomyces cerevisiae is presented. The model is based on rate equations for the individual reactions and aims to predict changes in the levels of intra- and extracellular metabolites after a glucose pulse, as described in part I of this study. Kinetic analysis focuses on a time scale of seconds, thereby neglecting biosynthesis of new enzymes. The model structure and experimental observations are related to the aerobic growth of the yeast. The model is based on material balance equations of the key metabolites in the extracellular environment, the cytoplasm and the mitochondria, and includes mechanistically based, experimentally matched rate equations for the individual enzymes. The model includes removal of metabolites from glycolysis and TCC for biosynthesis, and also compartmentation and translocation of adenine nucleotides. The model was verified by in vivo diagnosis of intracellular enzymes, which includes the decomposition of the network of reactions to reduce the number of parameters to be estimated simultaneously. Additionally, sensitivity analysis guarantees that only those parameters are estimated that contribute to systems trajectory with reasonable sensitivity. The model predictions and experimental observations agree reasonably well for most of the metabolites, except for pyruvate and adenine nucleotides. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 592-608, 1997.     

Arabidopsis indole synthase, a homolog of tryptophan synthase alpha, is an enzyme involved in the Trp-independent indole-containing metabolite biosynthesis

The plant tryptophan (Trp) biosynthetic pathway produces many secondary metabolites with diverse functions.Indole-3-acetic acid (IAA),proposed as a derivative from Trp or its precursors,plays an essential role in plant growth and development.Although the Trp-dependant and Trp-independent IAA biosynthetic pathways have been proposed,the enzymes,reactions and regulatory mechanisms are largely unknown.In Arabidopsis,indole-3-glycerol phosphate (IGP) is suggested to serve as a branchpoint component in the Trp-independent IAA biosynthesis.To address whether other enzymes in addition to Trp synthase α(TSA1) catalyze IGP cleavage,we identified and characterized an indole synthase (INS) gene,a homolog of TSA1 in Arabidopsis.INS exhibits different subcellular localization from TSA1 owing to the lack of chloroplast transit peptide (cTP).In silico data show that the expression levels of INS and TSA1 in all examined organs are quite different.Histochemical staining of INS promoter-GUS transgenic lines indicates that INS is expressed in vascular tissue of cotyledons,hypocotyls,roots and rosette leaves as well as in flowers and siliques.INS is capable of complementing the Trp auxotrophy of Escherichia coil △trpA strain,which is defective in Trp synthesis due to the deletion of TSA.This implies that INS catalyzes the conversion of IGP to indole and may be involved in the biosynthesis of Trp-independent IAA or other secondary metabolites in Arabidopsis.     

Structural enzymology of cholesterol biosynthesis and storage

Cholesterol biosynthesis occurs in the endoplasmic reticulum (ER). Its lego-like construction from water-soluble small metabolites via intermediates of increasing complexity to water-insoluble cholesterol requires numerous distinct enzymes. Dysfunction of the involved enzymes can cause several human inborn defects and diseases. Here, we review recent structures of three key cholesterol biosynthetic enzymes: Squalene epoxidase (SQLE), NAD(P)-dependent steroid dehydrogenase-like (NSDHL), and 3β-hydroxysteroid Δ⁸-Δ⁷ isomerase termed EBP. Moreover, we discuss structures of acyl-CoA:cholesterol acyltransferase (ACAT) enzymes, which are responsible for forming cholesteryl esters from cholesterol to maintain cholesterol homeostasis in the ER. The structures of these enzymes reveal their catalytic mechanism and provide a molecular basis to develop drugs for treating diseases linked to their dysregulation.     

Twin anchors of the soybean isoflavonoid metabolon: evidence for tethering of the complex to the endoplasmic reticulum by IFS and C4H

Isoflavonoids are specialized plant metabolites, almost exclusive to legumes, and their biosynthesis forms a branch of the diverse phenylpropanoid pathway. Plant metabolism may be coordinated at many levels, including formation of protein complexes, or ‘metabolons’, which represent the molecular level of organization. Here, we have confirmed the existence of the long‐postulated isoflavonoid metabolon by identifying elements of the complex, their subcellular localizations and their interactions. Isoflavone synthase (IFS) and cinnamate 4–hydroxylase (C4H) have been shown to be tandem P450 enzymes that are anchored in the ER, interacting with soluble enzymes of the phenylpropanoid and isoflavonoid pathways (chalcone synthase, chalcone reductase and chalcone isomerase). The soluble enzymes of these pathways, whether localized to the cytoplasm or nucleus, are tethered to the ER through interaction with these P450s. The complex is also held together by interactions between the soluble elements. We provide evidence for IFS interaction with upstream and non‐consecutive enzymes. The existence of such a protein complex suggests a possible mechanism for flux of metabolites into the isoflavonoid pathway. Further, through interaction studies, we identified several candidates that are associated with GmIFS2, an isoform of IFS, in soybean hairy roots. This list provides additional candidates for various biosynthetic and structural elements that are involved in isoflavonoid production. Our interaction studies provide valuable information about isoform specificity among isoflavonoid enzymes, which may guide future engineering of the pathway in legumes or help overcome bottlenecks in heterologous expression.     

Redox metabolism of glutathione in the red blood cell.

A theoretical study of the biochemical and clinical aspects of the reduction-oxidation metabolism of glutathione in the mature red blood cell is presented. A summarizing survey of the literature data has led to the formulation of a mathematical model which comprises the kinetic properties of the enzymes partaking in the hexose monophosphate pathway (HMP) and the oxidation of NADPH and GSH. The model takes the form of a system of differential equations describing the motion of metabolites in one cell. The interactions between metabolites ane enzymes, in particular between glutathione and the SH-dependent enzymes of glucose phosphorylation and HMP have been included into the model...