Guanine deaminase: Significance, assay, identification of inhibitors, and evidence for redox regulation

T Shaw, DS Bowden. Victorian Infectious Diseases Reference Laboratory y, Victoria 3051, Australia

For nearly half a century, the enzyme guanine deaminase (GDA; EC 3.5.4.3), has been recognized as a critical component of the enzymatic machinery of purine homeostasis in both prokaryotes and eukaryotes. GDA (also known as guanase, cypin and nedasin), which has been exceptionally highly conserved during evolution, is a small, zinc-dependent homodimer that catalyses the irreversible hydrolytic deamination of guanine, producing ammonium ions and xanthine. Four GDA isoforms, resulting from alternative mRNA splicing, have been identified. GDA is the main regulator of the intracellular guanine nucleotide pool, which is the smallest and most critical of the nucleotide pools in many cell types. Besides serving as cofactors and nucleic acid precursors, guanine nucleotides are essential components of numerous signalling pathways; they also regulate cell motility and sub-cellular trafficking via direct and indirect interactions with cytoskeletal proteins. GDA expression is developmentally regulated in mammals, in which it appears to control the supply of deoxyguanosine for mitochondrial DNA synthesis. In adult humans GDA expression is restricted mainly to hepatocytes, primitive immune cells and specific components of the nervous system, with lesser activity in some renal cells. GDA plays vital, linked roles in hepatic purine homeostasis, neural development and innate and acquired immunity. GDA activity in body fluids is barely detectable by most assays and its detection in serum or cerebrospinal fluid (CSF) is regarded as sensitive indicator of tissue damage or neoplasia. Increased neural GDA expression has been associated with stress, psychoses and depression.

Although GDA is an attractive prospective drug target for treatment of both liver diseases and cognitive disorders, very few effective inhibitors and/or activators of GDA have been reported to date, partly because of technical difficulties associated with assaying GDA activity: the enzyme is subject to regulation by numerous factors including redox state, divalent metal cations, certain anions and endogenous nucleotides as well as being inhibited by its own substrate and products, so its kinetics are complex. Bothe guanine and xanthine are barely soluble in aqueous media, which is also problematic.

We have developed an ultrasensitive high-throughput fluorometricmicrotitre plate assay for GDA by using coupled enzyme reactions (xanthine oxidase, uricase and peroxidase) that ultimately generate hydrogen peroxide, which is detected by oxidation of 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red). The high emission of the fluorescent product resorufin permits increases in sensitivity of the order of more than 1000-fold relative to conventional spectrophotometric and colorimetric assays. GDA purified from rabbit liver (purchased from Sigma) or partly purified from human sera collected during acute phase hepatitis A infection were used as the enzyme source. The assay protocol was essentially as described by Fernandez et al., [1], except that fluorimetric rather than colorimetric detection was used.Microtitre plate assays were performed at 37 °C and pH 7.6 in phosphate buffered saline containing 20 mM dimethylsulfone to increase the solubility of guanine and xanthine.

We have used the assay to monitor serum GDA as an indicator of liver damage and repair during the course of viral hepatitis and as an indicator of response to antiviral chemotherapy for chronic hepatitis B and C. We have also it to identify potential inhibitors of GDA and/or xanthine oxidase. Activities of GDA inhibitors identified by the fluorometric assay were confirmed using spectrophotometric assays in which guanine hydrolysis was monitored by decreases in absorbance at 245nm.

Besides the purine derivatives already reported by Fernández and colleaugues [1], we identified additional inhibitors with Kis in the micromolar range, the more potent of which included 8-hydroxyguanine (8-OH-G), a major product of oxidative damage to DNA and RNA. Its inhibitory activity against human serum GDA was comparable to that of the reference inhibitor 5-Aminoimidazole-4-carboxamide (Ki~3 microM) The enzyme Km for guanine in this assay was 6.0 +/- 0.1 microM, whilst 8-OH-G and some related purine derivatives had Kis < 5 microM.

Together with existing data, these observations suggest a further level of complexity in the function of GDA as a regulator of guanine nucleotide signalling and further emphasize its biological significance. The presence of probable binding sites for Sp1 and NF-kappaB in the promoter region of the GDA gene, together with circumstantial evidence from clinical observations, implies that transcription and expression of GDA is regulated by redox stress: our results suggest that 8-hydroxyguanine and 8-hydroxy-2’-deoxyguanosine may regulate the activity of the GDA in vivo.

Reference[1] Fernández, J.R.et al. Bioorg Med Chem. 2010; 18:6748-6755.