Adenylosuccinate lyase (adenylosuccinate adenosine 5′-monophosphate lyase, EC 22.214.171.124) was discovered in yeast and chick liver by Carter and Cohen,15 and by Buchanan's group.16 The enzyme is found in nearly all organisms and tissues examined.17– 22 A brief review of its properties is mostly restricted to the mammalian enzyme.
The two reactions catalyzed by ADSL involve the nonhydrolytic cleavage of the C-N bond linking the succinate moiety to the nucleotide part of the substrate, to yield fumarate (Fig. 112-2). Both eliminations are similar to that catalyzed by the urea cycle enzyme, argininosuccinate lyase.19 The conclusion that both SAICAR and S-AMP are cleaved by the same enzyme is based on the following evidence (reviewed in reference19): (a) the ratio of activities with both substrates remains the same along all purification procedures of the enzyme; (b) both substrates exhibit mutual competitive inhibition; and (c) mutant lower organisms which lack S-AMP cleavage activity are also unable to cleave SAICAR. ADSL displays hyperbolic kinetics. Km of both SAICAR and S-AMP is 1 to 10 μM for the enzyme of human erythrocytes,23 rat muscle,24 human cultured fibroblasts,25 and for human recombinant enzyme.26 The product of the two reactions, which it catalyzes, inhibits ADSL. Ki is 5 to 10 μM for AICAR and AMP, and 0.2 to 2.8 mM for fumarate.23,26,27 Competitive inhibition by AICAR and AMP, and noncompetitive inhibition by fumarate, indicate that ADSL from various sources, including yeast,28 murine cells,29 human erythrocytes,23 and the human recombinant enzyme,26 follows a simple, ordered, sequential reaction mechanism in which fumarate is the first product released, followed by the nucleotide. Intraperitoneal administration of AICAriboside, which is phosphorylated into AICAR by adenosine kinase,30 has been used to inhibit ADSL in skeletal muscle of rats in vivo. 31,32
ADSL is also capable of efficiently cleaving a variety of purine and nonpurine analogues, among which 6-mercaptopurinoadenylosuccinate,33 2′-deoxyadenylosuccinate, β-D-arabinosyladenylosuccinate,34 8-aza-adenylosuccinate and succino-4-aminopyrazo- lo(3,4-d)pyrimidine ribonucleotide, an allopurinol derivative,21 and 2′-3′-dideoxyadenylosuccinate.35 The latter is an intermediate in the conversion of the anti-HIV compounds 2′,3′-dideoxyadenosine and 2′,3′-dideoxyinosine into their active triphosphate derivatives.36 Other adenylosuccinate analogues are inhibitory of ADSL and have been investigated as potential antimetabolites (see below).
Studies of purified ADSL from human erythrocytes,23 rat liver,37 and muscle24 have shown that the native enzyme has a molecular weight of ≈200,000 and is composed of 4 subunits. Nucleotide-predicted amino acid sequences of ADSL have been reported for a number of tissues, including chicken liver,38 human liver,39 and mouse kidney.40 From open cDNA reading frames of 1377 nucleotides, a sequence of 459 amino acids was deduced for both the chicken and human enzyme. However, the high degree of identity of the nucleotide sequence upstream of the originally reported initiation codon of the cDNAs cloned from both sources with the mouse sequence,40 and correction from C to A of the third nucleotide of the human sequence,41 have revealed a first ATG, 75 nucleotides 5′ of the originally reported initiation codon. The open reading frame thus comprises 1452 nucleotides and encodes a protein that is 25 amino acids longer at the N-terminus, containing 484 amino acids. At the amino acid level, human ADSL was found 85 percent identical to the chicken enzyme,39 and 94 percent identical to the murine enzyme.40
ADSL also bears sequence homology to a group of enzymes that generate fumarate from different substrates, namely argininosuccinate lyase, aspartase, and class II fumarases. Moreover, it displays sequence similarity with δ-crystallin, the major structural component of the lenses of most birds and reptiles.42– 44 This transparent protein is considered to have evolved from argininosuccinate lyase by gene sharing,45 because it has retained its enzymic activity. Most highly conserved among the similar regions of the enzymes cited above is an 11-amino acid span (amino acids 288 to 298; GSSAMPYKRNP in the corrected sequence of human ADSL), which is considered the “fumarate lyase” signature.38 (See also Chap. 85.)
The catalytic mechanism of ADSL has been shown to involve a general base catalyst, removing a proton from the succinyl group of the substrate, acting in concert with a general acid catalyst that protonates the amino group left on AICAR or AMP.46,47 Several phylogenetically conserved amino acid residues have been proposed to perform these functions.26,38 Affinity labeling of Bacillus subtilis ADSL with 4-bromo-2,3-dioxobutyl thio derivatives of AMP has led to the suggestion that a highly conserved Arg112 is involved in the binding of the substrate by the enzyme, and that another highly conserved residue, His141, corresponding to His159 in human ADSL, may be the general base catalyst48– 50 and that His68, corresponding to His86 in the human enzyme, may be the general acid catalyst (Lee et al 1998, 1999). Construction of homology models of Bacillus subtilis ADSL as a guide to selection of site-directed mutagenesis candidates has shown that His89 is also required for catalysis (Brosius and Colman, 2000) and that Lys268 and Glu275 are critical for enzyme function (Brosius and Colman, 2002).
Crystal structures of several enzymes of this superfamily of lyases have been solved, namely those of turkey δ-crystallin,43 Escherichia coli fumarase,44 aspartase (Shi et al, 1997), argininosuccinate lyase (Turner et al, 1997), and ADSL. The latter has been crystallized from B. subtilis,51 Pyrobaculum aerophilum (Toth et al, 2000), and Thermotoga maritima (Toth and Yeates, 2000). All crystal structures reveal tetramers with four active-site clefts, each formed by the intersection of residues from three separate subunits of the tetramer. Manual placement of both substrates of ADSL in the active site suggests that no special adaptation by the protein is necessary to accommodate them since the succinyl moiety of SAICAR can rotate in the same orientation as that of S-AMP (Toth and Yeates, 2000).
Tissue Distribution and Isozymes
In man, ADSL activity has been measured in erythrocytes,1,23,52 granulocytes, lymphocytes, liver, kidney, and muscle.3,53 ADSL remains active in cultured mammalian cells, including fibroblasts3,25,53– 55 and lymphoblasts.56 A number of earlier observations had suggested that isoforms of ADSL exist. Whereas starvation induced a profound, approximately 90 percent, decrease of the activity of ADSL in rat liver and spleen, it had no effect on the activity of the enzyme in muscle, brain, and kidney.20 Isoelectric focusing of rat muscle ADSL showed the existence of three isomeric forms present in similar amounts.57 A reevaluation of the effect of starvation on rat liver ADSL has, however, revealed that the decreased activity recorded under this condition could be nearly completely suppressed by the addition of protease inhibitors in the homogenization buffers (Maliekal et al, 2002). Accordingly, ADSL mRNA decreased by no more than 20 percent after 4 days of starvation. Nevertheless, a number of observations have suggested the occurrence of alternative splicing. Expression of cDNA clones in an epidermoid carcinoma and two colon carcinoma cell lines revealed two ADSL mRNAs with different sizes: 1.8 and 2.5 kb.58 Northern blots of mouse tissues,40 revealed only a single predominant 1.9-kb message, but in chicken liver,38 an abundant mRNA of approximately 1.7 kb was accompanied by two minor messages of 1.2 and 3.0 kb. In humans, two ADSL mRNAs are formed by alternative splicing of exon 12 and are expressed in several tissues, including liver, muscle, kidney, and brain (Kmoch et al, 2000). Deletion of the 177-bp exon 12 provokes the loss of 59 amino acids and results in an inactive enzyme protein. Although, studies in patients with ADSL deficiency (see below) show that the activity of the enzyme is lost to a different extent in various tissues, and is normal in others, these findings can probably not be explained by the existence of isozymes of ADSL.
Adenylosuccinate Lyase in Cancer Cells
Up to threefold elevated activities of ADSL as compared to normal are found in a variety of tumor cells, particularly those derived from liver and kidney.59,60 These increases appear specific for neoplasia insofar as they are not seen in regenerating liver. Kinetic properties of the enzyme of a rapidly growing hepatoma were similar to those of normal ADSL.60 Increases in liver ADSL activity were also noted as early as 48 to 72 h following the administration of hepatocarcinogens such as 3′-methyl-4-dimethylaminobenzene and thioacetamide.61 In experimentally induced rat tumor models, elevation of ADSL activity was a reliable early indicator of the presence of hepatic or breast tumor.62 In human breast and prostate tumors, a high activity of ADSL was also an indicator of malignancy.63 On the other hand, differentiation of colon carcinoma cells has been shown to be accompanied by downregulation of the expression of ADSL.58
Because enhancement of the de novo synthesis of purines is one of the main characteristics of tumor cells, and because ADSL intervenes twice in this process, the potential exists for use of inhibitors of ADSL as antimetabolites. Adenylophosphonopropionate, in which the aspartate moiety of S-AMP is replaced by 3-phosphoalanine, is the most potent inhibitor (Ki ≈ 0.02 μM) of the enzyme reported sofar.64 Yet, owing to their multiple negative charges, both adenylophosphonopropionate and its nucleoside fail to penetrate cells.65 The fluorinated derivatives of aspartate, threo-β-fluoroaspartate and erythro-β-fluoroaspartate, also potently inhibit ADSL after their conversion into adenylosuccinate and SAICAR derivatives by adenylosuccinate synthetase and SAICAR synthetase, respectively.24,66 However, both compounds are highly toxic because fluoroaspartate can substitute for aspartate in other pathways, most notably protein synthesis.67 Alanosine, an investigational anticancer drug, can substitute for aspartate in the SAICAR synthetase reaction, forming alanosyl-carboxylateaminoimidazole ribotide (alanosyl-CAIR), which acts mainly as an inhibitor of adenylosuccinate synthetase, but which also exerts an inhibitory effect on ADSL.68