The Concise Guide to Pharmacology 2013/14: Enzymes

The Concise Guide to PHARMACOLOGY 2013/14 provides concise overviews of the key properties of over 2000 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.12444/full. Enzymes are one of the seven major pharmacological targets into which the Guide is divided, with the others being G protein-coupled receptors, ligand-gated ion channels, 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. A new landscape format has easy to use tables comparing related targets. It is a condensed version of material contemporary to late 2013, which is presented in greater detail and constantly updated on the website www.guidetopharmacology.org, superseding data presented in previous Guides to Receptors and Channels. 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 the Guide to Receptors and Channels, providing a permanent, citable, point-in-time record that will survive database updates.


Comments:
With the exception of mitochondrial 5′nucleotidase, each of the 5′-nucleotidases are localised to the cytoplasm.

Carboxylases and decarboxylases Carboxylases
Overview: The carboxylases allow the production of new carbon-carbon bonds by introducing HCO3or CO2 into target molecules. Two groups of carboxylase activities, some of which are bidirectional, can be defined on the basis of the cofactor requirement, making use of biotin (EC 6 Loss-of-function mutations in γ-glutamyl carboxylase are associated with clotting disorders

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 L-DOPA, 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, (-

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 subse-quently reduced 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 galac-tosylceramides. 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
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.

Sphingomyelin synthase
Overview: Following translocation from the ER to the Golgi under the influence of the ceramide transfer protein, sphingomyelin synthases allow the formation of sphingomyelin by the transfer of phosphocholine from the phospholipid phosphatidylcholine.
Sphingomyelin synthase-related protein 1 is structurally related but lacks sphingomyelin synthase activity.  [57] Glycoceramides are an extended family of sphingolipids, differing in the content and organization of the sugar moieties, as well as the acyl sidechains

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

Comment
The enzyme is associated with the plasma membrane [74] --

Comments:
Two further structurally-related proteins have been identified (ASAH2B, P0C7U1 and ASAH2C, P0C7U2). ASAH2B appears to be an enzymatically inactive protein, which may result from gene duplication and truncation.

Alkaline ceramidases
Overview: The five 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: A ceramide kinase-like protein has been identified in the human genome (CERKL, Q49MI3).

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).

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 endogenously by cAMP and with some pharmacological selectivity by 8-pCPT-2'-O-Me-cAMP [90]. Once activated, Epacs induce an enhanced activity of the monomeric G proteins, Rap1 and Rap2 by facilitating binding of GTP in place of GDP, leading to activation of phospholipase C [126].

Nomenclature
Comments: 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 cGMP specific and is activated by the α-subunit of transducin (Gαt) 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.
-.-, were originally defined by their strong absorbance at 450 nm due to the reduced carbon monoxide-complexed haem component of the cytochromes. They are an extensive family of haem-containing monooxygenases with a huge range of both endogenous and exogenous substrates. Listed below are the human enzymes; their relationship with rodent CYP450 enzyme activities is obscure in that the species orthologue may not mediate metabolism of the same substrates. Although the majority of CYP450 enzyme activities are concentrated in the liver, the extrahepatic enzyme activities also contribute to patho/ physiological processes. Genetic variation of CYP450 isoforms is widespread and likely underlies a significant proportion of the individual variation to drug administration.

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 conju-gation 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 non-enzymatic manner, leading to suggestions for their use as biomarkers of oxidative stress. Molecular targets for their action have yet to be defined.

Leukotriene and lipoxin metabolism
Overview: Leukotriene A4 (LTA4), produced by 5-LOX activity, and lipoxins may be subject to further oxidative metabolism; ω-hydroxylation is mediated by CYP4F2 and CYP4F3, while β-oxidation in mitochondria and peroxisomes proceeds in a manner dependent on coenzyme A conjugation. Conjugation of LTA4 at the 6 position with reduced glutathione to generate LTC4 occurs under the influence of leukotriene C4 synthase, with the subsequent formation of LTD4 and LTE4, all three of which are agonists at CysLT receptors. LTD4 formation is catalysed by γ-glutamyltransferase, and subsequently dipeptidase 2 removes the terminal glycine from LTD4 to generate LTE4. Leukotriene A4 hydrolase converts the 5,6-epoxide LTA4 to the 5-hydroxylated LTB4, an agonist for BLT receptors. LTA4 is also acted upon by 12S-LOX to produce the trihydroxyeicosatetraenoic acids lipoxins LXA4 and LXB4. Treatment with a LTA4 hydrolase inhibitor in a murine model of allergic airway inflammation increased LXA4 levels, in addition to reducing LTB4, in lung lavage fluid [186].

Endocannabinoid turnover
Overview: The principle endocannabinoids are 2arachidonoylglycerol (2AG) and anandamide (Narachidonoylethanolamine, AEA), thought to be generated on demand rather than stored. 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 involved is diacylglycerol lipase (DGL), whilst several routes for anandamide synthesis have been described, the best characterized of which involves N-acylphosphatidylethanolamine-phospholipase D (NAPE-PLD, [206]). Inactivation of these endocannabinoids appears to occur predominantly through monoacylglycerol lipase (MGL) and fatty acid amide hydrolase (FAAH) for 2arachidonoylglycerol and anandamide, respectively. Note that these enzymes also contribute to the turnover of many endogenous ligands inactive at CB1 and CB2 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 [195,198,207].

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 [213] 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 GABAA or GABAB receptors and may be accumulated 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.

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).

Phosphoinositide-specific phospholipase C
Overview: Phosphoinositide-specific phospholipase C (PLC, EC 3.1.4.11) catalyses the hydrolysis of PIP2 to IP3 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 Gq/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-ε1 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-δ activity. PLC has been suggested to be activated non-selectively by the small molecule m3M3FBS [218], although this mechanism of action has been questioned [235]. The aminosteroid U73122 has been described as an inhibitor of phosphoinositide-specific PLC [257], although its selectivity among the isoforms is untested and it has been reported to occupy the H1 histamine receptor [230]. Overview: Phospholipase A2 (PLA2, EC 3.1.1.4) cleaves the sn-2 fatty acid of phospholipids, primarily phosphatidylcholine, to generate lysophosphatidylcholine and arachidonic acid. Most commonlyused inhibitors (e.g. BEL, ATFMK or MAFP) are either non-selective within the family of phospholipase A2 enzymes or have activity against other eicosanoid-metabolising enzymes. A binding protein for secretory phospholipase A2 has been identified which shows modest selectivity for sPLA2-1B over sPLA2-2A, and also binds snake toxin phospholipase A2 [216]. The binding protein appears to have clearance function for circulating secretory phospholipase A2, as well as signalling functions, and is a candidate antigen for idiopathic membraneous nephropathy [219].

Phosphatidylinositol phosphate kinases
Overview: PIP2 is generated by phosphorylation of PI

Hydrogen sulfide synthesis
Overview: Hydrogen sulfide is a putative gasotransmitter, with similarities to NO and carbon monoxide. Although the enzymes indicated have multiple enzymatic activities, the focus here is the generation of hydrogen sulfide and the enzymatic characteristics are described accordingly. Cystathionine β-synthase and cystathionine γ-lyase are pyridoxal phosphate-dependent enzymes, while L-cysteine:2-oxoglutarate aminotransferase and 3-mercaptopyruvate sulfurtransferase function in combination as a pyridoxal phosphate-independent pathway.

Lanosterol biosynthesis pathway
Overview: Lanosterol is a precursor for cholesterol, which is synthesized primarily in the liver in a pathway often described as the mevalonate or HMG-CoA reductase pathway. The first two steps (formation of acetoacetyl CoA and the mitochondrial generation of HMG-CoA) are also associated with oxidation of fatty acids.

Peptidases and proteinases
Overview: Peptidases and proteinases hydrolyse peptide bonds, and can be simply divided on the basis of whether terminal peptide bonds are cleaved (exopeptidases and exoproteinases) at the amino terminus (aminopeptidases) or carboxy terminus It is beyond the scope of the Guide to list all peptidase and proteinase activities; this summary focuses on selected enzymes of significant pharmacological interest.

Cysteine (C) Peptidases: Caspases
Overview: Caspases, (E.C. 3.4.22.-) which derive their name from Cysteine ASPartate-specific proteASES, include at least two families; initiator caspases (caspases 2, 8, 9 and 10), which are able to hydrolyse and activate a second family of effector cas-pases (caspases 3, 6 and 7), which themselves are able to hydrolyse further cellular proteins to bring about programmed cell death. Caspases are heterotetrameric, being made up of two pairs of subunits, generated by a single gene product, which is proteo-lysed to form the mature protein. Members of the mammalian inhibitors of apoptosis proteins (IAP) are able to bind the procaspases, thereby preventing maturation to active proteinases. Hip-His Leu has been used experimentally as a probe for ACE1. ACE1 appears to express a distinct GPI hydrolase activity [296].

Nomenclature
Abz-Ser-Pro-Tyr(NO2)-OH has been used experimentally as a probe for ACE2

ADAMTS metallopeptidases
Overview: ADAMTS (with thrombospondin motifs) metalloproteinases cleave cell-surface or transmembrane proteins to generate soluble and membrane-limited products. suggests the presence of 518 protein kinases in man, with over 100 protein kinase-like pseudogenes [342]. It is beyond the scope of the Guide to list all these protein kinase activities; this summary focuses on AGC protein kinases associated with GPCR signalling, which may be divided into 15 subfamilies in man.

Nomenclature
Most inhibitors of these enzymes have been assessed in cell-free investigations and so may appear to 'lose' potency and selectivity in intact cell assays. In particular, ambient ATP concentrations may be influential in responses to inhibitors, since the majority are directed at the ATP binding site [319].

G protein-coupled receptor kinases
Overview: G protein-coupled receptor kinases, epitomized by βARK, are involved in the rapid phosphorylation and desensitization of GPCR. Classically, high concentrations of β2-adrenoceptor agonists binding to the receptor lead to the consequent activation and dissociation of the heterotrimeric G protein Gs. Gαs activates adenylyl cyclase activity, while Gβγ subunits perform other functions, one of which is to recruit βARK to phosphorylate serine/threonine residues in the cytoplasmic tail of the β2-adrenoceptor. The phosphorylated receptor binds, with high affinity, a member of the arrestin family (ENSFM00250000000572), which prevents further signalling through the G protein (uncoupling) and may allow interaction with scaffolding proteins, such as clathrin, with the possible consequence of internalization and/or degradation.

Protein kinase A
Overview: Cyclic AMP-mediated signalling involves regulation of cyclic nucleotide-gated ion channels, members of the Rap guanine nucleotide exchange family (Epac, ENSFM00250000000899) and activation of protein kinase A (PKA, also known as cyclic AMP-dependent protein kinase). PKA is a heterotetrameric enzyme composed of two regulatory and two catalytic subunits, which can be distinguished from Epac (exchange protein directly activated by cAMP, [320]) by differential activation by N 6 benzyl-cAMP (see Table)  Comments: Other members of the PKA family are PRKX (X-linked protein kinase, PKX1, P51817) and PRKY (Y-linked protein kinase, PRKY, O43930). PRKX and PRKY are expressed on X and Y chromosomes, respectively, and appear to interchange in some XX males and XY females [347].
Classical protein kinase C isoforms: PKCα, PKCβ, PKCγ. Members of the classical protein kinase C family are activated by Ca 2+ and diacylglycerol, and may be inhibited by GF109203X, calphostin C, Gö6983, chelerythrine and Ro318220.
Nomenclature protein kinase C, alpha protein kinase C, beta protein kinase C, gamma

Sphingosine 1-phosphate turnover
Overview: S1P (sphingosine 1-phosphate) is a pro-survival signal, in contrast to ceramide. It is formed by the sphingosine kinase-catalysed phosphorylation of sphingosine. S1P can be released from cells to act as an agonist at a family of five G protein-coupled receptors (S1P1-5) but also has intracellular targets. S1P can be dephosphorylated back to sphingosine or hydrolysed to form hexadecanal and phosphoethanolamine. Sphingosine choline phosphotransferase (EC 2.7.8.10) generates sphingosylphosphocholine from sphingosine and CDP-choline. Sphingosine β-galactosyltransferase (EC 2.4.1.23) generates psychosine from sphingosine in the presence of UDP-α-Dgalactose. The molecular identities of these enzymes have not been confirmed.