Parameter Reliability and Understanding Enzyme Function.

Knowledge of the Michaelis-Menten parameters and their meaning in different circumstances is an essential prerequisite to understanding enzyme function and behaviour. The published literature contains an abundance of values reported for many enzymes. The problem concerns assessing the appropriateness and validity of such material for the purpose to which it is to be applied. This review considers the evaluation of such data with particular emphasis on the assessment of its fitness for purpose.

6-Hydroxydopamine: a far from simple neurotoxin.

6-Hydroxydopamine (6-OHDA), which is a neurotoxin that selectively destroys catecholaminergic nerves in sympathetically innervated tissues, has been used to provide a model of Parkinson's disease in experimental animals. It is rapidly autoxidised to yield potentially toxic products and reactive oxygen species. Its ability to release Fe(II) from protein storage sites also results in the formation of hROS. This account will consider how this family of toxic products may contribute to the observed effects of 6-OHDA.

A mechanism for bistability in glycosylation.

Glycosyltransferases are a class of enzymes that catalyse the posttranslational modification of proteins to produce a large number of glycoconjugate acceptors from a limited number of nucleotide-sugar donors. The products of one glycosyltransferase can be the substrates of several other enzymes, causing a combinatorial explosion in the number of possible glycan products. The kinetic behaviour of systems where multiple acceptor substrates compete for a single enzyme is presented, and the case in which high concentrations of an acceptor substrate are inhibitory as a result of abortive complex formation, is shown to result in non-Michaelian kinetics that can lead to bistability in an open system. A kinetic mechanism is proposed that is consistent with the available experimental evidence and provides a possible explanation for conflicting observations on the ß-1,4-galactosyltransferases. Abrupt switching between steady states in networks of glycosyltransferase-catalysed reactions may account for the observed changes in glycosyl-epitopes in cancer cells.

90 years of monoamine oxidase: some progress and some confusion.

It would not be practical to attempt to deal with all the advances that have informed our understanding of the behavior and functions of this enzyme over the past 90 years. This account concentrates key advances that explain why the monoamine oxidases remain of pharmacological and biochemical interest and on some areas of continuing uncertainty. Some issues that remain to be understood or are in need of further clarification are highlighted.

A Knowledge-Based System for Display and Prediction of O-Glycosylation Network Behaviour in Response to Enzyme Knockouts.

O-linked glycosylation is an important post-translational modification of mucin-type protein, changes to which are important biomarkers of cancer. For this study of the enzymes of O-glycosylation, we developed a shorthand notation for representing GalNAc-linked oligosaccharides, a method for their graphical interpretation, and a pattern-matching algorithm that generates networks of enzyme-catalysed reactions. Software for generating glycans from the enzyme activities is presented, and is also available online. The degree distributions of the resulting enzyme-reaction networks were found to be Poisson in nature. Simple graph-theoretic measures were used to characterise the resulting reaction networks. From a study of in-silico single-enzyme knockouts of each of 25 enzymes known to be involved in mucin O-glycan biosynthesis, six of them, ß-1,4-galactosyltransferase (ß4Gal-T4), four glycosyltransferases and one sulfotransferase, play the dominant role in determining O-glycan heterogeneity. In the absence of ß4Gal-T4, all Lewis X, sialyl-Lewis X, Lewis Y and Sda/Cad glycoforms were eliminated, in contrast to knockouts of the N-acetylglucosaminyltransferases, which did not affect the relative abundances of O-glycans expressing these epitopes. A set of 244 experimentally determined mucin-type O-glycans obtained from the literature was used to validate the method, which was able to predict up to 98% of the most common structures obtained from human and engineered CHO cell glycoforms.

Assessment of Enzyme Inhibition: A Review with Examples from the Development of Monoamine Oxidase and Cholinesterase Inhibitory Drugs.

The actions of many drugs involve enzyme inhibition. This is exemplified by the inhibitors of monoamine oxidases (MAO) and the cholinsterases (ChE) that have been used for several pharmacological purposes. This review describes key principles and approaches for the reliable determination of enzyme activities and inhibition as well as some of the methods that are in current use for such studies with these two enzymes. Their applicability and potential pitfalls arising from their inappropriate use are discussed. Since inhibitor potency is frequently assessed in terms of the quantity necessary to give 50% inhibition (the IC50 value), the relationships between this and the mode of inhibition is also considered, in terms of the misleading information that it may provide. Incorporation of more than one functionality into the same molecule to give a multi-target-directed ligands (MTDLs) requires careful assessment to ensure that the specific target effects are not significantly altered and that the kinetic behavior remains as favourable with the MTDL as it does with the individual components. Such factors will be considered in terms of recently developed MTDLs that combine MAO and ChE inhibitory functions.

Reflections on 60 years of publication of the Journal of Neurochemistry.

This review reflects on the origins, development, publishing trends, and scientific directions of the Journal of Neurochemistry over its 60 year lifespan as seen by key contributors to the Journal's production. The Journal first appeared in May 1956 with just two issues published in that inaugural year. By 1963, it appeared monthly and, by 2002, 24 hard copy issues were published yearly. In 2014, the Journal became online only. For much of its time, the Journal was managed through two separate editorial offices each with their respective Chief Editor (the 'Western' and 'Eastern' hemispheres). The Journal was restructured to operate through a single editorial office and Editor-in-Chief from 2013. Scientifically, the Journal progressed through distinct scientific eras with the first two decades generally centered around developments in methodology followed by a period when publications delved deeper into underlying mechanisms. By the late 1980s, the Journal had entered the age of genetics and beyond, with an increasing focus on neurodegenerative diseases. Reviews have played a regular part in the success of J Neurochem with focused special and virtual issues being a highlight of recent years. Today, 60 years and onwards, J Neurochem continues to be a leading source of top-quality, original and review articles in neuroscience. We look forward to its continued success at the forefront of neurochemistry in the decades to come. This article celebrates 60 years of publication of Journal of Neurochemistry including personal reminiscences from some of the Chief Editors, past and present, as well as input from some of the key contributors to the Journal over this period. We highlight the scientific, technological, and publishing developments along the way, with reference to key papers published in the Journal. The support of the Journal toward the aims and objectives of the International Society for Neurochemistry (ISN) is also emphasized. This article is part of the 60th Anniversary special issue.

Galactosyltransferase 4 is a major control point for glycan branching in N-linked glycosylation.

Protein N-glycosylation is a common post-translational modification that produces a complex array of branched glycan structures. The levels of branching, or antennarity, give rise to differential biological activities for single glycoproteins. However, the precise mechanism controlling the glycan branching and glycosylation network is unknown. Here, we constructed quantitative mathematical models of N-linked glycosylation that predicted new control points for glycan branching. Galactosyltransferase, which acts on N-acetylglucosamine residues, was unexpectedly found to control metabolic flux through the glycosylation pathway and the level of final antennarity of nascent protein produced in the Golgi network. To further investigate the biological consequences of glycan branching in nascent proteins, we glycoengineered a series of mammalian cells overexpressing human chorionic gonadotropin (hCG). We identified a mechanism in which galactosyltransferase 4 isoform regulated N-glycan branching on the nascent protein, subsequently controlling biological activity in an in vivo model of hCG activity. We found that galactosyltransferase 4 is a major control point for glycan branching decisions taken in the Golgi of the cell, which might ultimately control the biological activity of nascent glycoprotein.

Characterization of the in vitro binding and inhibition kinetics of primary amine oxidase/vascular adhesion protein-1 by glucosamine.

BACKGROUND: Primary-amine oxidase (PrAO) catalyzes the oxidative deamination of endogenous and exogenous primary amines and also functions, in some tissues, as an inflammation-inducible endothelial factor, known as vascular adhesion protein-1. VAP-1 mediates the slow rolling and adhesion of lymphocytes to endothelial cells in a number of inflammatory conditions, including inflammation of the synovium. METHODS: Glucosamine binding to the enzyme was assessed spectrofluorometrically and the kinetics of inhibition of PrAO were determined spectrophotometrically through the use of direct or coupled assays, in the presence of different substrates. RESULTS: Glucosamine is not a substrate for PrAO, but acts as a time-dependent inhibitor of PrAO activity, displaying mixed inhibition kinetics. The observed inhibition and binding were augmented in the presence of H(2)O(2). CONCLUSIONS: Significant in vitro effects on PrAO require glucosamine in the millimolar concentration range and it is not clear at this stage whether a low but persistent level of PrAO inhibition might contribute to the anti-arthritic response. GENERAL SIGNIFICANCE: This work was aimed at characterizing the interactions of PrAO/VAP-1 with glucosamine, a widely used "over-the-counter" supplement for the treatment of osteoarthritis.

Enzyme nomenclature and classification: the state of the art.

The IUBMB enzyme classification system, available at the IUBMB ExplorEnz website, uses a four-component number (the EC number) that identifies an enzyme in terms of reaction catalysed. There were originally six recognized groups of enzymes: Oxidoreductases (EC 1), Transferases (EC 2), Hydrolases (EC 3), Lyases (EC 4), Isomerases (EC 5) and Ligases (EC 6). Of these, the lyases, which are defined as 'enzymes that cleave C-C, C-O, C-N and other bonds by means other than by hydrolysis or oxidation', present particular recognition and classification problems. Recently, a new class, the Translocases (EC 7), has been added, which incorporates enzymes that catalyse the movement of ions or molecules across membranes or their separation within membranes. A new subclass of the isomerases has also been included for those enzymes that alter the conformations of proteins and nucleic acids. Newly reported enzymes are being regularly added to the list after validation and where new information affects the classification of an existing entry, a new EC number is created, but the old one is not reused.

Highly reactive oxygen species: detection, formation, and possible functions.

The so-called reactive oxygen species (ROS) are defined as oxygen-containing species that are more reactive than O(2) itself, which include hydrogen peroxide and superoxide. Although these are quite stable, they may be converted in the presence of transition metal ions, such as Fe(II), to the highly reactive oxygen species (hROS). hROS may exist as free hydroxyl radicals (HO·), as bound ("crypto") radicals or as Fe(IV)-oxo (ferryl) species and the somewhat less reactive, non-radical species, singlet oxygen. This review outlines the processes by which hROS may be formed, their damaging potential, and the evidence that they might have signaling functions. Since our understanding of the formation and actions of hROS depends on reliable procedures for their detection, particular attention is given to procedures for hROS detection and quantitation and their applicability to in vivo studies.

Interaction of L-lysine and soluble elastin with the semicarbazide-sensitive amine oxidase in the context of its vascular-adhesion and tissue maturation functions.

The copper-containing quinoenzyme semicarbazide-sensitive amine oxidase (EC 1.4.3.21; SSAO) is a multifunctional protein. In some tissues, such as the endothelium, it also acts as vascular-adhesion protein 1 (VAP-1), which is involved in inflammatory responses and in the chemotaxis of leukocytes. Earlier work had suggested that lysine might function as a recognition molecule for SSAO/VAP-1. The present work reports the kinetics of the interaction of L-lysine and some of its derivatives with SSAO. Binding was shown to be saturable, time-dependent but reversible and to cause uncompetitive inhibition with respect to the amine substrate. It was also specific, since D-lysine, L-lysine ethyl ester and epsilon-acetyl-L-lysine, for example, did not bind to the enzyme. The lysine-rich protein soluble elastin bound to the enzyme relatively tightly, which may have relevance to the reported roles of SSAO in maintaining the extracellular matrix (ECM) and in the maturation of elastin. Our data show that lysyl residues are not oxidized by SSAO, but they bind tightly to the enzyme in the presence of hydrogen peroxide. This suggests that binding in vivo of SSAO to lysyl residues in physiological targets might be regulated in the presence of H(2)O(2), formed during the oxidation of a physiological SSAO substrate, yet to be identified.

Clinically available iron chelators induce neuroprotection in the 6-OHDA model of Parkinson's disease after peripheral administration.

The iron content of the substantia nigra pars compacta increases in the brains of Parkinson's disease patients. Hence, its removal by iron chelators may retard the progression of the disease. However, information on the ability of clinically available iron chelators to cross the blood brain barrier and be neuroprotective is limited. In this present study three iron chelators, which are currently approved for clinical use, namely the hexadendate, deferrioxamine, the bidentate deferiprone and the tridendate chelator deferasirox have been investigated for their efficacy to induce neuroprotection. Previous studies have shown that both deferiprone and deferrioxamine exert neuroprotection in the 6-hydroxy dopamine (6-OHDA) model but no such studies have investigated deferasirox. Focal administration of deferasirox (0.5, 2 and 10 µg) into the substantia nigra pars compacta of rats significantly attenuated the loss of dopaminergic neurons and striatal dopamine content resulting from 6-OHDA toxicity. Systemic administration of deferasirox (20 mg/kg), deferiprone (10 mg/kg) or deferrioxamine (30 mg/kg), to the 6-OHDA rat model of Parkinson's disease, significantly attenuated the loss of dopaminergic neurons and striatal dopamine content. Further studies to comprehend the action of these chelators showed that local application of either 0.4 mM deferrioxamine, or 1 mM deferasirox, via a microdialysis probe into the striatum, prior to that of 200 µM 6-OHDA, prevented the generation of hydroxyl radicals. Our results confirm that the administration of these chelators show therapeutic efficacy and should be considered as therapeutic agents for the treatment of Parkinson's disease.

ExplorEnz: the primary source of the IUBMB enzyme list.

ExplorEnz is the MySQL database that is used for the curation and dissemination of the International Union of Biochemistry and Molecular Biology (IUBMB) Enzyme Nomenclature. A simple web-based query interface is provided, along with an advanced search engine for more complex Boolean queries. The WWW front-end is accessible at http://www.enzyme-database.org, from where downloads of the database as SQL and XML are also available. An associated form-based curatorial application has been developed to facilitate the curation of enzyme data as well as the internal and public review processes that occur before an enzyme entry is made official. Suggestions for new enzyme entries, or modifications to existing ones, can be made using the forms provided at http://www.enzyme-database.org/forms.php.

Tracing metabolic pathways from enzyme data.

The IUBMB Enzyme List is widely used by other databases as a source for avoiding ambiguity in the recognition of enzymes as catalytic entities. However, it was never designed for activities such as pathway tracing, which have become increasingly important in systems biology. This is because it often relies on generic or representative reactions to show the reactions catalysed by enzymes of wide specificity. It is necessary to go to databases such as BRENDA to find further, more detailed, information on what is known about the range of substrates for any particular enzyme. In order to provide a framework for tracing pathways involving any specific enzyme or metabolite, we have created a Reactions Database from the material in the Enzyme List. This allows reactions to be searched by substrate/product and pathways to be traced from any selected starting/seed substrate. An extensive synonym glossary allows searches by many of the alternative names, including accepted abbreviations, by which a chemical compound may be known. This database was necessary for the development of the application Reaction Explorer (http://www.reaction-explorer.org), which was written in REALbasic to search the Reactions Database and draw metabolic pathways from reactions selected by the user. Having input the name of the starting compound (the "seed"), the user is presented with a list of all reactions containing that compound and then selects the product of interest as the next point on the ensuing graph. The pathway diagram is then generated as the process iterates. A contextual menu is provided, which allows the user to (i) remove a compound from the graph, along with all associated links; (ii) search the reactions database again for additional reactions involving the compound and (iii) search for the compound within the Enzyme List.

Degradation of thymic humoral factor γ2 in human, rat and mouse blood: An experimental and theoretical study.

The degradation of the immunomodulatory octapeptide, thymic humoral factor γ2 (THF-γ2, thymoctonan) has been studied in whole blood samples from human, rat and mouse. The peptide, Leu-Glu-Asp-Gly-Pro-Lys-Phe-Leu, was shown to be rapidly degraded by peptidases. The half-life of the intact peptide was less than 6 min at 37 °C in blood from the three species tested. The main fragments formed from THF-γ2 were found to be Glu-Asp-Gly-Pro-Lys-Phe-Leu (2-8), Asp-Gly-Pro-Lys-Phe-Leu (3-8) and Glu-Asp-Gly-Pro-Lys (2-6) in human and in rat blood and 2-8 and 2-6 in mouse blood. Analysis of the time course of degradation revealed a sequential removal of single amino acids from the N-terminus (aminopeptidase activities) in a process that was apparently unable to cleave the Gly-Pro bond (positions 4-5 in the peptide) together with an independent cleavage of the Lys-Phe bond (positions 6-7 in the peptide) to release the dipeptide Phe-Leu. This behaviour and the effects of inhibitors showed the involvement of metallo-exopeptidases in the N-terminal digestion and a phosphoramidon-sensitive metallo-endopeptidase in the cleavage of the Lys-Phe bond. The degradation patterns in human blood were modelled in terms of the competing pathways involved approximating to first-order kinetics, and an analytical solution obtained via the method of Laplace Transforms. The half-life of THF degradation in whole rat blood sample was found to be significantly lower than in human or mouse.

The application of bathophenanthroline for the determination of free iron in parallel with hROS in microdialysis samples.

BACKGROUND: Free, non-protein bound, Fe(II), which can catalyse the formation of the toxic highly-reactive oxygen species (hROS), has been implicated in several neurodegenerative conditions. The determination of free Fe(II) and Fe(III) in samples obtained from microdialysis experiments has been limited by the small amounts of sample available. NEW METHOD: This work describes the development of a HPLC, with absorbance detection, method, based on the complexation of Fe(II) with bathophenanthroline disulfonate (BS), which allows a complete extracellular iron analysis with the small sample amounts that are available from in vivo microdialysis in rat brain. RESULTS: Microdialysis experiments using 6-hydroxydopamine stimulation, showed that basal-as well as evoked levels of extracellular Fe(II) and total iron could be determined in parallel with measurements of hROS formation. COMPARISON WITH EXISTING METHODS: Although a spectrophotometric BS-based assay has been reported for use in microdialysis samples from large animals, the present procedure is applicable to the small sample sizes available from studies in rat brain. It is simpler than the alternative, involving inductively-coupled plasma mass spectrometry. CONCLUSIONS: The procedure described is simple and sensitive, giving a linear response in the Fe(II) concentration range of 50 -2000 nM. A 20 min microdialysis sample (flow-rate 3 µl/min) yields sufficient material for triplicate determinations of the evoked release of Fe(II) and total iron whilst leaving sufficient sample volume for determining hROS and amine or amino-acid neurotransmitter release.

Human aldehyde dehydrogenase genes: alternatively spliced transcriptional variants and their suggested nomenclature.

OBJECTIVE: The human aldehyde dehydrogenase (ALDH) gene superfamily consists of 19 genes encoding enzymes critical for NAD(P)-dependent oxidation of endogenous and exogenous aldehydes, including drugs and environmental toxicants. Mutations in ALDH genes are the molecular basis of several disease states (e.g. Sjögren-Larsson syndrome, pyridoxine-dependent seizures, and type II hyperprolinemia) and may contribute to the etiology of complex diseases such as cancer and Alzheimer's disease. The aim of this nomenclature update was to identify splice transcriptional variants principally for the human ALDH genes. METHODS: Data-mining methods were used to retrieve all human ALDH sequences. Alternatively spliced transcriptional variants were determined based on (i) criteria for sequence integrity and genomic alignment; (ii) evidence of multiple independent cDNA sequences corresponding to a variant sequence; and (iii) if available, empirical evidence of variants from the literature. RESULTS AND CONCLUSION: Alternatively spliced transcriptional variants and their encoded proteins exist for most of the human ALDH genes; however, their function and significance remain to be established. When compared with the human genome, rat and mouse include an additional gene, Aldh1a7, in the ALDH1A subfamily. To avoid confusion when identifying splice variants in various genomes, nomenclature guidelines for the naming of such alternative transcriptional variants and proteins are recommended herein. In addition, a web database (www.aldh.org) has been developed to provide up-to-date information and nomenclature guidelines for the ALDH superfamily.

Theobromine and related methylxanthines as inhibitors of Primary Amine Oxidase.

Methylxanthines are among the most widely consumed drugs in the world and evidence of their health benefits has been growing in recent years. Primary Amine Oxidase (PrAO) has been recognized as a therapeutic target for the amelioration of inflammatory, vascular, and neurodegenerative diseases. Previous work in our laboratories showed that caffeine inhibited Bovine PrAO with a Ki of 1.0 mM using benzylamine as substrate. This study aimed to extend our previous work and explore the possibility that related methylxanthines might influence PrAO activity. While paraxanthine, theophylline, and 7-methylxanthine had little effect on PrAO, theobromine was a noncompetitive inhibitor with a Ki of 276 ± 44 µM. The specific structural elements of methylxanthines that are required for inhibition allow us to suggest that their binding site on PrAO may be a target for therapeutics. The health benefits associated with dietary methylxanthine consumption could involve PrAO inhibition. PRACTICAL APPLICATIONS: Inhibition of PrAO by methylxanthines may be significant in conferring health benefits. The design of PrAO inhibitors based on the structural motifs identified in this study (N-methylation at specific locations) is indicated. Existing therapeutics based on a core xanthine structure can be evaluated for their effects on PrAO. PrAO inhibition must be considered as a potential mediator of the beneficial health effects of some methylxanthines. If inhibition in human tissues is comparable to, or greater than, that found in these studies it points to an important role for these compounds in human health.

Partial inhibition of complex I activity increases Ca-independent glutamate release rates from depolarized synaptosomes.

Mitochondria have been implicated in the pathogenesis of several neurodegenerative disorders and, in particular, complex I (NADH:ubiquinone oxidoreductase, EC 1.6.5.3) activity has been shown to be partially reduced in postmortem studies of the substantia nigra of Parkinson's disease patients. The present study examines the effect of partial inhibition of complex I activity on glutamate release from rat brain synaptosomes. Following a 40% inhibition of complex I activity with rotenone, it was found that Ca(2+)-independent release of glutamate increased from synaptosomes depolarized with 4-aminopyridine. Highest rates of glutamate release were found to occur between 60-90% complex I inhibition. A similar pattern of increase was shown to occur in synaptosomes depolarized with KCl. The increase in glutamate release was found to correlate to a significant decrease in ATP. Inhibition of complex I activity by 40% was also shown to cause a significant collapse in mitochondrial membrane potential (Deltapsi(m)). These results suggest that partial inhibition of complex I activity in in situ mitochondria is sufficient to significantly increase release of glutamate from the pre-synaptic nerve terminal. The relevance of these results in the context of excitotoxicity and the pathogenesis of neurodegenerative disorders is discussed.