The Concise Guide to PHARMACOLOGY 2015/16: Enzymes

The Concise Guide to PHARMACOLOGY 2015/16 provides concise overviews of the key properties of over 1750 human drug targets with their pharmacology, plus links to an open access knowledgebase of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. The full contents can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.13354/full. G protein‐coupled receptors are one of the eight major pharmacological targets into which the Guide is divided, with the others being: G protein‐coupled receptors, ligand‐gated ion channels, voltage‐gated ion channels, other ion channels, nuclear hormone receptors, catalytic receptors and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The Concise Guide is published in landscape format in order to facilitate comparison of related targets. It is a condensed version of material contemporary to late 2015, which is presented in greater detail and constantly updated on the website www.guidetopharmacology.org, superseding data presented in the previous Guides to Receptors & Channels and the Concise Guide to PHARMACOLOGY 2013/14. It is produced in conjunction with NC‐IUPHAR and provides the official IUPHAR classification and nomenclature for human drug targets, where appropriate. It consolidates information previously curated and displayed separately in IUPHAR‐DB and GRAC and provides a permanent, citable, point‐in‐time record that will survive database updates.

monophosphatase only in the presence of high substrate concentrations. Some inhibitors are irreversible, including a group known as suicide substrates, which bind to the ligand recognition site and then couple covalently to the enzyme. It is beyond the scope of the Guide to give mechanistic information about the inhibitors described, although generally this information is avail-able from the indicated literature. Many enzymes require additional entities for functional activity. Some of these are used in the catalytic steps, while others promote a particular conformational change. Co-factors are tightly bound to the enzyme and include metal ions and heme groups. Co-enzymes are typically small molecules which accept or donate functional groups to assist in the enzymatic reaction. Examples include ATP, NAD, NADP and S-adenosylmethionine, as well as a number of vitamins, such as riboflavin (vitamin B1) and thiamine (vitamin B2). Where co-factors/co-enzymes have been identified, the Guide indicates their involvement.

Enzymes
Peptidases and proteinases AD: Aspartic (A) Peptidases A22: Presenilin Overview: Presenilin (PS)-1 or -2 act as the catalytic component/essential co-factor of the γ-secretase complex responsible for the final carboxy-terminal cleavage of amyloid precursor protein (APP) [249] in the generation of amyloid beta (Aβ) [6,471]. Given that the accumulation and aggregation of Aβ in the brain is pivotal in the development of Alzheimer's disease (AD), inhibition of PS activity is one mechanism being investigated as a therapeutic option for AD [177]. Several small molecule inhibitors of PS-1 have been investigated, with some reaching early clinical trials, but none have been formally approved. Comment: folate hydrolase is also known as NAALADase as it is responsible for the hydrolysis of N-acetaspartylglutamate to form N-acetylaspartate and L-glutamate. In the gut, the enzyme assists in the assimilation of folate by hydrolysing dietary poly-gamma-glutamylfolate. The enzyme is highly expressed in the prostate, and its expression is up-regulated in cancerous tissue. A tagged version of the antibody capromab has been used for imaging purposes.

Enzymes Acetylcholine turnover
Overview: Acetylcholine is familiar as a neurotransmitter in the central nervous system and in the periphery. In the somatic nervous system, it activates nicotinic acetylcholine receptors at the skeletal neuromuscular junction. It is also employed in the autonomic nervous system, in both parasympathetic and sympathetic branches; in the former, at the smooth muscle neuromuscu-lar junction, activating muscarinic acetylcholine receptors. In the latter, acetylcholine is involved as a neurotransmitter at the ganglion, activating nicotinic acetylcholine receptors. Acetylcholine is synthesised in neurones through the action of choline Oacetyltransferase and metabolised after release through the extracellular action of acetylcholinesterase and cholinesterase. Choline is accumulated from the extracellular medium by selective transporters (see SLC5A7 and the SLC44 family). Acetylcholine is accumulated in synaptic vesicles through the action of the vesicular acetylcholine transporter SLC18A3. Comments Splice variants of choline O-acetyltransferase are suggested to be differentially distributed in the periphery and CNS (see [28]).

--
Comments: A number of organophosphorus compounds inhibit acetylcholinesterase and cholinesterase irreversibly, including pesticides such as chlorpyrifos-oxon, and nerve agents such as tabun, soman and sarin. AChE is unusual in its exceptionally high turnover rate which has been calculated at 740 000/min/molecule [505].

Enzymes
Adenosine turnover Overview: A multifunctional, ubiquitous molecule, adenosine acts at cell-surface G protein-coupled receptors, as well as numerous enzymes, including protein kinases and adenylyl cyclase. Extracellular adenosine is thought to be produced either by export or by metabolism, predominantly through ecto-5'-nucleotidase activity (also producing inorganic phosphate). It is inactivated either by extracellular metabolism via adenosine deaminase (also producing ammonia) or, following uptake by nucleoside trans-porters, via adenosine deaminase or adenosine kinase (requiring ATP as co-substrate). Intracellular adenosine may be produced by cytosolic 5'-nucleotidases or through S-adenosylhomocysteine hydrolase (also producing L-homocysteine).

L-Arginine turnover
Enzymes L-Arginine turnover Overview: L-arginine is a basic amino acid with a guanidino sidechain. As an amino acid, metabolism of L-arginine to form L-ornithine, catalysed by arginase, forms the last step of the urea production cycle. L-Ornithine may be utilised as a precursor of polyamines (see Carboxylases and Decarboxylases) or recycled via L-argininosuccinic acid to L-arginine. L-Arginine may itself be decarboxylated to form agmatine, although the prominence of this pathway in human tissues is uncertain. L-Arginine may be used as a precursor for guanidoacetic acid formation in the creatine synthesis pathway under the influence of arginine:glycine amidinotransferase with L-ornithine as a byproduct. Nitric oxide synthase uses L-arginine to generate nitric oxide, with L-citrulline also as a byproduct. L-Arginine in proteins may be subject to post-translational mod-ification through methylation, catalysed by protein arginine methyltransferases. Subsequent proteolysis can liberate asymmetric N G ,N G -dimethyl-L-arginine (ADMA), which is an endogenous inhibitor of nitric oxide synthase activities. ADMA is hydrolysed by dimethylarginine dimethylhydrolase activities to generate L-citrulline and dimethylamine. Overview: Arginase (EC 3.5.3.1) are manganese-containing isoforms, which appear to show differential distribution, where the ARG1 isoform predominates in the liver and erythrocytes, while ARG2 is associated more with the kidney.

Nitric oxide synthases
Enzymes L-Arginine turnover Nitric oxide synthases Overview: Nitric oxide synthases (NOS, E.C. 1.14.13.39) utilise L-arginine (not D-arginine) and molecular oxygen to generate nitric oxide and L-citrulline. The nomenclature suggested by NC-IUPHAR of NOS I, II and III [340] has not gained wide accep-tance. eNOS and nNOS isoforms are activated at concentrations of calcium greater than 100 nM, while iNOS shows higher affinity for Ca 2+ /calmodulin (CALM1 CALM2 CALM3, P62158) and thus appears to be constitutively active. All the three isoforms are ho-modimers and require sapropterin, flavin adenine dinucleotide, flavin mononucleotide and NADPH for catalytic activity. L-NAME is an inhibitor of all three isoforms, with an IC 50 value in the micromolar range. 3-bromo-7NI (pIC 50 6.1-6.5) [40], 7NI (pIC 50

5.3) [19]
Comments: The reductase domain of NOS catalyses the reduction of cytochrome c and other redox-active dyes [322]. NADPH:O 2 oxidoreductase catalyses the formation of superoxide anion/H 2 O 2 in the absence of L-arginine and sapropterin. Loss-of-function mutations in γ-glutamyl carboxylase are associated with clotting disorders.

Carboxylases and decarboxylases
Comments: Dicarboxylic acids including citric acid are able to activate ACC1/ACC2 activity allosterically. PCC is able to function in forward and reverse modes as a ligase (carboxylase) or lyase (decarboxylase) activity, respectively. Loss-of-function mutations in GGCX are associated with clotting disorders.

Enzymes Carboxylases and decarboxylases Decarboxylases
Overview: The decarboxylases generate CO 2 and the indicated products from acidic substrates, requiring pyridoxal phosphate or pyruvic acid as a co-factor. The presence of a functional ADC activity in human tissues has been questioned [89].
AADC is a homodimer. L-aspartic acid is a less rapidly metabolised substrate of mouse brain glutamic acid decarboxylase generating β-alanine [530]. Autoantibodies against GAD1 and GAD2 are elevated in type 1 diabetes mellitus and neurological disorders (see Further reading).
L-aspartic acid is a less rapidly metabolised substrate of mouse brain glutamic acid decarboxylase generating β-alanine [530]. The activity of ODC is regulated by the presence of an antizyme (ENSG00000104904) and an ODC antizyme inhibitor (ENSG00000155096).

Further Reading
Bale

Catecholamine turnover
Enzymes Catecholamine turnover Overview: Catecholamines are defined by the presence of two adjacent hydroxyls on a benzene ring with a sidechain containing an amine. The predominant catacholamines in mammalian biology are the neurotransmitter/hormones dopamine, (-)-noradrenaline (norepinephrine) and (-)-adrenaline (epinephrine).
These hormone/transmitters are synthesized by sequential metabolism from L-phenylalanine via L-tyrosine. Hydroxylation of L-tyrosine generates levodopa, which is decarboxylated to form dopamine. Hydroxylation of the ethylamine sidechain generates (-)-noradrenaline (norepinephrine), which can be methylated to form (-)-adrenaline (epinephrine). In particular neuronal and adrenal chromaffin cells, the catecholamines dopamine, (-)-noradrenaline and (-)-adrenaline are accumulated into vesicles under the influence of the vesicular monoamine transporters (VMAT1/SLC18A1 and VMAT2/SLC18A2). After release into the synapse or the blood-stream, catecholamines are accumulated through the action cell-surface transporters, primarily the dopamine (DAT/SLC6A3) and norepinephrine transporter (NET/SLC6A2). The primary routes of metabolism of these catecholamines are oxidation via monoamine oxidase activities of methylation via catechol Omethyltransferase.

Ceramide turnover
Enzymes Ceramide turnover Overview: Ceramides are a family of sphingophospholipids synthesized in the endoplasmic reticulum, which mediate cell stress responses, including apoptosis, autophagy and senescence, Serine palmitoyltransferase generates 3-ketosphinganine, which is reduced to sphinganine (dihydrosphingosine). N-Acylation allows the formation of dihydroceramides, which are subsequently re-duced to form ceramides. Once synthesized, ceramides are trafficked from the ER to the Golgi bound to the ceramide transfer protein, CERT (COL4A3BP, Q9Y5P4). Ceramide can be metabolized via multiple routes, ensuring tight regulation of its cellular levels. Addition of phosphocholine generates sphingomyelin while carbohydrate is added to form glucosyl-or galactosylceramides.
Ceramidase re-forms sphingosine or sphinganine from ceramide or dihydroceramide. Phosphorylation of ceramide generates ceramide phosphate. The determination of accurate kinetic parameters for many of the enzymes in the sphingolipid metabolic pathway is complicated by the lipophilic nature of the substrates.

Ceramide synthase
Enzymes Ceramide turnover Ceramide synthase Overview: This family of enzymes, also known as sphingosine N-acyltransferase, is located in the ER facing the cytosol with an as-yet undefined topology and stoichiometry. Ceramide synthase in vitro is sensitive to inhibition by the fungal derived toxin, fumonisin B1.

Enzymes
Ceramide turnover Acid ceramidase Overview: The six human ceramidases may be divided on the basis of pH optimae into acid, neutral and alkaline ceramidases, which also differ in their subcellular location.

Neutral ceramidases
Enzymes Ceramide turnover Neutral ceramidases Overview: The six human ceramidases may be divided on the basis of pH optimae into acid, neutral and alkaline ceramidases, which also differ in their subcellular location.

Comments
The enzyme is associated with the plasma membrane [475].

-
Comments: ASAH2B appears to be an enzymatically inactive protein, which may result from gene duplication and truncation.

Enzymes
Ceramide turnover Alkaline ceramidases Overview: The six human ceramidases may be divided on the basis of pH optimae into acid, neutral and alkaline ceramidases, which also differ in their subcellular location.

Enzymes
Chromatin modifying enzymes

Overview:
Chromatin modifying enzymes, and other chromatin-modifying proteins, fall into three broad categories: writers, readers and erasers. The function of these proteins is to dynamically maintain cell identity and regulate processes such as differentiation, development, proliferation and genome integrity via recognition of specific 'marks' (covalent post-translational modifications) on histone proteins and DNA [267]. In normal cells, tissues and organs, precise co-ordination of these proteins ensures expression of only those genes required to specify phenotype or which are required at specific times, for specific functions. Chromatin modifications allow DNA modifications not coded by the DNA sequence to be passed on through the genome and underlies heritable phenomena such as X chromosome inactivation, aging, heterochromatin formation, reprogramming, and gene silencing (epigenetic control). To date at least eight distinct types of modifications are found on histones. These include small covalent modifications such as acetylation, methylation, and phosphorylation, the attachment of larger modifiers such as ubiquitination or sumoylation, and ADP ribosylation, proline isomerization and deimination. Chromatin modifications and the functions they regulate in cells are reviewed by Kouzarides (2007) [267]. Writer proteins include the histone methyltransferases, histone acetyltransferases, some kinases and ubiquitin ligases. Readers include proteins which contain methyl-lysinerecognition motifs such as bromodomains, chromodomains, tudor domains, PHD zinc fingers, PWWP domains and MBT domains. Erasers include the histone demethylases and histone deacetylases (HDACs and sirtuins). Dysregulated epigenetic control can be associated with human diseases such as cancer [129], where a wide variety of cellular and pro-tein abberations are known to perturb chromatin structure, gene transcription and ultimately cellular pathways [24,439]. Due to the reversible nature of epigenetic modifications, chromatin regulators are very tractable targets for drug discovery and the development of novel therapeutics. Indeed, small molecule inhibitors of writers (e.g. azacitidine and decitabine target the DNA methyltransferases DNMT1 and DNMT3 for the treatment of myelodysplastic syndromes [165,520]) and erasers (e.g. the HDAC inhibitors vorinostat, romidepsin and belinostat for the treatment of T-cell lymphomas [144,254]) are already being used in the clinic. The search for the next generation of compounds with improved specificity against chromatin-associated proteins is an area of intense basic and clinical research [56]. Current progress in this field is reviewed by Simó-Riudalbas and Esteller (2015) [440]. They generate both mono-methylated and dimethylated products; these may be symmetric (SDMA) or asym-metric (N G ,N G -dimethyl-L-arginine) versions, where both guanidine nitrogens are monomethylated or one of the two is dimethylated, respectively.

-Histone deacetylases (HDACs)
Overview: Histone deacetylases act as erasers of epigenetic acetylation marks on lysine residues in histones. Removal of the acetyl groups facilitates tighter packing of chromatin (heterochromatin formation) leading to transcriptional repression. The histone deacetylase family has been classified in to five subfamilies based on phylogenetic comparison with yeast homologues: Class I contains HDACs 1, 2, 3 and 8 Class IIa contains HDACs 4, 5, 7 and 9 Class IIb contains HDACs 6 and 10 Class III contains the sirtuins (SIRT1-7) Class IV contains only HDAC11. Classes I, II and IV use Zn + as a co-factor, whereas catalysis by Class III enzymes requires NAD + as a co-factor, and members of this subfamily have ADP-ribosylase activity in addition to protein deacetylase function [420].

Enzymes
Cyclic nucleotide turnover Overview: Cyclic nucleotides are second messengers generated by cyclase enzymes from precursor triphosphates and hydrolysed by phosphodiesterases. The cellular actions of these cyclic nucleotides are mediated through activation of protein kinases (cAMP-and cGMP-dependent protein kinases), ion channels (cyclic nucleotide-gated, CNG, and hyperpolarization and cyclic nucleotide-gated, HCN) and guanine nucleotide exchange factors (GEFs, Epac). Comments: Nitric oxide has been proposed to inhibit AC5 and AC6 selectively [210], although it is unclear whether this phenomenon is of physiological significance. A soluble adenylyl cyclase has been described (ADCY10, Q96PN6 [48]), unaffected by either Gα or Gβγ subunits, which has been suggested to be a cytoplasmic bicarbonate (pH-insensitive) sensor [75]. It can be inhibited selectively by KH7 (pIC 50 5.0-5.5) [208].

Exchange protein activated by cyclic AMP (Epac)
Enzymes Cyclic nucleotide turnover Exchange protein activated by cyclic AMP (Epac) Overview: Epacs are members of a family of guanine nucleotide exchange factors (ENSFM00250000000899), which also includes RapGEF5 (GFR, KIAA0277, MR-GEF, Q92565) and RapGEFL1 (Link-GEFII, Q9UHV5). They are activated endoge-nously by cyclic AMP and with some pharmacological selectivity by 8-pCPT-2'-O-Me-cAMP [126]. Once activated, Epacs induce an enhanced activity of the monomeric G proteins, Rap1 and Rap2 by facilitating binding of guanosine-5'-triphosphate in place of guanosine 5'-diphosphate, leading to activation of phospholipase C [423]. PDE6 is a membrane-bound tetramer composed of two catalytic chains (PDE6A or PDE6C and PDE6B), an inhibitory chain (PDE6G or PDE6H) and the PDE6D chain. The enzyme is essentially cyclic GMP specific and is activated by the α-subunit of transducin (Gα t ) and inhibited by sildenafil, zaprinast and dipyridamole with potencies lower than those observed for PDE5A. Defects in PDE6B are a cause of retinitis pigmentosa and congenital stationary night blindness. Responsible for -hydroxylation of LTB 4 , LXB 4 [335], and polyunsaturated fatty acids [135,194] Converts PGH 2 to 19-hydroxyPGH 2 [ Comments Inhibited by dazoxiben [398] and camonagrel [182].

Overview:
The principle endocannabinoids are 2-arachidonoylglycerol (2AG) and anandamide (Narachidonoylethanolamine, AEA), thought to be generated on demand rather than stored, although this may not always be the case [10]. Mechanisms for release and re-uptake of endocannabinoids (and related entities) are unclear, although candidates for intracellular transport have been suggested. For the generation of 2-arachidonoylglycerol, the key enzyme in-volved is diacylglycerol lipase (DGL), whilst several routes for anandamide synthesis have been described, the best characterized of which involves N-acylphosphatidylethanolaminephospholipase D (NAPE-PLD, [438]). Inactivation of these endocannabinoids appears to occur predominantly through monoacylglycerol lipase (MGL) and fatty acid amide hydrolase (FAAH) for 2-arachidonoylglycerol and anandamide, respectively. Note that these enzymes also contribute to the turnover of many endogenous ligands inactive at CB 1 and CB 2 cannabinoid receptors, such as N-oleoylethanolamide, N-palmitoylethanolamine and 2-oleoyl glycerol. In vitro experiments indicate that the endocannabinoids are also substrates for oxidative metabolism via cyclooxygenase, lipoxygenase and cytochrome P450 enzyme activities [9,145,450].

Nomenclature
Diacylglycerol lipase α Diacylglycerol lipase β N-Acylphosphatidylethanolamine-phospholipase D  [171] anandamide oleamide N-oleoylethanolamide N-palmitoylethanolamine [518] oleamide N-oleoylethanolamide anandamide N-palmitoylethanolamine [518] N-palmitoylethanolamine MEA SEA N-oleoylethanolamide anandamide [495]  Comments: Many of the compounds described as inhibitors are irreversible and so potency estimates will vary with incubation time. FAAH2 is not found in rodents [518] and few of the inhibitors described have been assessed at this enzyme activity.

Further Reading
Blankman

Enzymes Eicosanoid turnover
Overview: Eicosanoids are 20-carbon fatty acids, where the usual focus is the polyunsaturated analogue arachidonic acid and its metabolites. Arachidonic acid is thought primarily to derive from phospholipase A2 action on membrane phosphatidylcholine, and may be re-cycled to form phospholipid through con-jugation with coenzyme A and subsequently glycerol derivatives. Oxidative metabolism of arachidonic acid is conducted through three major enzymatic routes: cyclooxygenases; lipoxygenases and cytochrome P450-like epoxygenases, particularly CYP2J2. Isoprostanes are structural analogues of the prostanoids (hence the nomenclature D-, E-, F-isoprostanes and isothromboxanes), which are produced in the presence of elevated free radicals in a nonenzymatic manner, leading to suggestions for their use as biomarkers of oxidative stress. Molecular targets for their action have yet to be defined.

Enzymes
Eicosanoid turnover Leukotriene and lipoxin metabolism Overview: Leukotriene A 4 (LTA 4 ), produced by 5-LOX activity, and lipoxins may be subject to further oxidative metabolism; -hydroxylation is mediated by CYP4F2 and CYP4F3, while βoxidation in mitochondria and peroxisomes proceeds in a manner dependent on coenzyme A conjugation. Conjugation of LTA 4 at the 6 position with reduced glutathione to generate LTC 4 occurs under the influence of leukotriene C 4 synthase, with the subsequent formation of LTD 4 and LTE 4 , all three of which are agonists at CysLT receptors. LTD 4 formation is catalysed by γglutamyltransferase, and subsequently dipeptidase 2 removes the terminal glycine from LTD 4 to generate LTE 4 . Leukotriene A 4 hydrolase converts the 5,6-epoxide LTA 4 to the 5-hydroxylated LTB 4 , an agonist for BLT receptors. LTA 4 is also acted upon by 12S-LOX to produce the trihydroxyeicosatetraenoic acids lipoxins LXA 4 and LXB 4 . Treatment with a LTA 4 hydrolase inhibitor in a murine model of allergic airway inflammation increased LXA 4 levels, in addition to reducing LTB 4 , in lung lavage fluid [400]. LTA 4 hydrolase is also involved in biosynthesis of resolvin Es. Aspirin has been reported to increase endogenous formation of 18S-hydroxyeicosapentaenoate (18S-HEPE) compared with 18R-HEPE, a resolvin precursor. Both enantiomers may be metabolised by human recombinant 5-LOX; recombinant LTA 4 hydrolase converted chiral 5S(6)-epoxide-containing intermediates to resolvin E1 and 18S-resolvin E1 [358].

GABA turnover
Enzymes GABA turnover Overview: The inhibitory neurotransmitter γ-aminobutyrate (GABA, 4-aminobutyrate) is generated in neurones by glutamic acid decarboxylase. GAD1 and GAD2 are differentially expressed during development, where GAD2 is thought to subserve a trophic role in early life and is distributed throughout the cytoplasm. GAD1 is expressed in later life and is more associated with nerve terminals [128] where GABA is principally accumulated in vesicles through the action of the vesicular inhibitory amino acid transporter SLC32A1. The role of γ-aminobutyraldehyde dehydrogenase (ALDH9A1) in neurotransmitter GABA synthesis is less clear. Following release from neurons, GABA may interact with either GABA A or GABA B receptors and may be accumu-lated in neurones and glia through the action of members of the SLC6 family of transporters. Successive metabolism through GABA transaminase and succinate semialdehyde dehydrogenase generates succinic acid, which may be further metabolized in the mitochondria in the tricarboxylic acid cycle. -Selective inhibitors s-allylglycine ---Comments L-aspartic acid is a less rapidly metabolised substrate of mouse brain glutamic acid decarboxylase generating β-alanine [530]. Autoantibodies against GAD1 and GAD2 are elevated in type 1 diabetes mellitus and neurological disorders (see Further reading).

Enzymes Glycerophospholipid turnover
Overview: Phospholipids are the basic barrier components of membranes in eukaryotic cells divided into glycerophospholipids (phosphatidic acid, phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylinositol and its phosphorylated derivatives) and sphingolipids (ceramide phosphorylcholine and ceramide phosphorylethanolamine).

Overview:
Phosphatidylinositol may be phosphorylated at either 3-or 4-positions on the inositol ring by PI 3-kinases or PI 4-kinases, respectively.

Enzymes
Glycerophospholipid turnover Phosphoinositide-specific phospholipase C Overview: Phosphoinositide-specific phospholipase C (PLC, EC 3.1.4.11), catalyses the hydrolysis of PIP 2 to IP 3 and 1,2diacylglycerol, each of which have major second messenger functions. Two domains, X and Y, essential for catalytic activity, are conserved in the different forms of PLC. Isoforms of PLC-β are activated primarily by G protein-coupled receptors through members of the G q/11 family of G proteins. The receptor-mediated activation of PLC-γ involves their phosphorylation by receptor tyrosine kinases (RTK) in response to activation of a variety of growth factor receptors and immune system receptors. PLC-1 may represent a point of convergence of signalling via both G protein-coupled and catalytic receptors. Ca 2+ ions are required for catalytic activity of PLC isoforms and have been suggested to be the major physiological form of regulation of PLC-AE activity. PLC has been suggested to be activated non-selectively by the small molecule m3M3FBS [21], although this mechanism of action has been questioned [271]. The aminosteroid U73122 has been described as an inhibitor of phosphoinositide-specific PLC [447], although its selectivity among the isoforms is untested and it has been reported to occupy the H1 histamine receptor [222]. A binding protein for secretory phospholipase A 2 has been identified which shows modest selectivity for sPLA 2 -1B over sPLA 2 -2A, and also binds snake toxin phospholipase A 2 [12]. The binding protein appears to have clearance function for circulating secretory phospholipase A 2 , as well as signalling functions, and is a candidate antigen for idiopathic membraneous nephropathy [27]. PLA 2 -G7 and PAFAH2 also express platelet-activating factor acetylhydrolase activity (EC 3.1.1.47).

Enzymes
Glycerophospholipid turnover Phosphatidylcholine-specific phospholipase D Overview: Phosphatidylcholine-specific phospholipase D (PLD, EC 3.1.1.4) catalyses the formation of phosphatidic acid from phosphatidylcholine. In addition, the enzyme can make use of alcohols, such as butanol in a transphosphatidylation reaction [399].

Enzymes Hydrolases
Overview: Listed in this section are hydrolases not accumulated in other parts of the Concise Guide, such as monoacylglycerol lipase and acetylcholinesterase. Pancreatic lipase is the predominant mechanism of fat digestion in the alimentary system; its inhibition is associated with decreased fat absorption. CES1 is present at lower levels in the gut than CES2 (P23141), but predominates in the liver, where it is responsible for the hydrolysis of many aliphatic, aromatic and steroid esters. Hormone-sensitive lipase is also a relatively non-selective esterase associated with steroid ester hydrolysis and triglyceride metabolism, particularly in adipose tissue. Endothelial lipase is secreted from endothelial cells and regulates circulating cholesterol in high density lipoproteins. Comments: In vitro analysis suggested IP 3 and IP 4 were poor substrates for SKIP, synaptojanin 1 and synaptojanin 2, but suggested that PIP 2 and PIP 3 were more efficiently hydrolysed [422]. Comments THI (2-Acetyl-5-tetrahydroxybutyl imidazole) inhibits the enzyme activity in intact cell preparations [426].

Further Reading
Bigaud

Enzymes
Thyroid hormone turnover

Overview:
The thyroid hormones triiodothyronine and thyroxine, usually abbreviated as triiodothyronine and T 4 , respectively, are synthesized in the thyroid gland by sequential metabolism of tyrosine residues in the glycosylated homodimeric protein thyroglobulin (TG, P01266) under the influence of the haem-containing protein iodide peroxidase. Iodide peroxidase/TPO is a haem-containing enzyme, from the same structural family as eosinophil peroxidase (EPX, P11678), lactoperoxidase (LPO, P22079) and myeloperoxidase (MPO, P05164). Circulating thyroid hormone is bound to thyroxine-binding globulin (SERPINA7, P05543).
Hydroxylated HIFs are then targeted for proteasomal degradation via the von Hippel-Lindau ubiquitination complex [232]. Under hypoxic conditions, the hydroxylation reaction is blunted which results in decreased HIF degradation. The surviving HIFs are then available to translocate to the nucleus where they heterodimerize with HIF-1β, effecting increased expression of hypoxia-inducible genes.
HIF-PH enzymes are being investigated as pharmacological targets as their inhibition mimics the hypoxic state and switches on transcription of genes associated with processes such as erythropoiesis and vasculogenesis [142]. Small molecule HIF-PH inhibitors are in clinical trial as novel therapies for the amelioration of anemia associated with chronic kidney disease [46].
Nomenclature egl-9 family hypoxia-inducible factor 2 egl-9 family hypoxia-inducible factor 1 egl-9 family hypoxia-inducible factor 3 Overview: The Poly ADP-ribose polymerase family is a series of enzymes, where the best characterised members are nuclear proteins which are thought to function by binding to single strand breaks in DNA, allowing the recruitment of repair enzymes by the synthesis of NAD-derived ADP-ribose polymers, which are subsequently degraded by a glycohydrolase (PARG, Q86W56).
Nomenclature poly (ADP-ribose) polymerase 1 poly (ADP-ribose) polymerase 2 poly (ADP-ribose) polymerase 3  [66]. Protein farnesyltransferase catalyses the post-translational formation of a thioether linkage between the C-1 of an isoprenyl group and a cysteine residue fourth from the C-terminus of a protein (ie to the CaaX motif, where 'a' is an aliphatic amino acid and 'X' is usually serine, me-thionine, alanine or glutamine; leucine for EC 2.5.1.59) [156]. Farnesyltransferase is a dimer, composed of an alpha and beta subunit and requires Mg 2+ and Zn 2+ ions as cofactors. The active site is located between the subunits. Prenylation creates a hydrophobic domain on protein tails which acts as a membrane anchor. Substrates of the prenyltransferases include Ras, Rho, Rab, other Ras-related small GTP-binding proteins, G-protein γ-subunits, nuclear lamins, centromeric proteins and many proteins involved in visual signal transduction. In relation to the causative association between oncogenic Ras proteins and cancer, farnesyltransferase has become an important mechanistic drug discovery target.

RAS subfamily
Enzymes 3.6.5.2 Small monomeric GTPases RAS subfamily Overview: The RAS proteins (HRAS, NRAS and KRAS) are small membrane-localised G protein-like molecules of 21 kd. They act as an on/off switch linking receptor and non-receptor tyrosine kinase activation to downstream cytoplasmic or nuclear events. Binding of GTP activates the switch, and hydrolysis of the GTP to GDP inactivates the switch. The RAS proto-oncogenes are the most frequently mutated class of proteins in human cancers. Common mutations compromise the GTP-hydrolysing ability of the proteins causing constitutive activation [457], which leads to increased cell proliferation and decreased apoptosis [550]. Because of their importance in oncogenic transformation these proteins have become the targets of intense drug discovery effort [23].