AMP deaminase

The enzyme adenylic acid deaminase catalyzes the deamination of AMP to IMP and ammonia. For the HPLC method, the assay involves the separation of the substrate, AMP, from the reaction product IMP. The enzyme is found in muscle. 

In the assay of Jahngen and Rossomando (1984), AMP and IMP were separated by means of ion-paired reverse-phased HPLC on a Q8 (/uBondapak) column with a mobile phase of 65 mM potassium phosphate (pH 3.6), 1 mM tetra-n-butylammonium phosphate, and 4% methanol. The column was eluted isocratically, and the eluent was monitored at 254 nm. When the formycin analogs were used, detection was at 295 nm at this wavelength there was no 

The reaction mixture contained, in a final volume of 250 fiL, imidazole-HC1 (pH 6.8) as the buffer, either 250 nmol AMP or the formycin analog formycin 5 -AMP (FoMP) as substrate, and the activators ATP and KCl. The reaction was started by the addition of the enzyme, and at intervals samples were withdrawn from the reaction tube and injected directly onto the HPLC column for analysis. 

The activity was prepared from the microorganism Dictyostelium dis-coideunt. Cells were lysed, and an S-100 fraction was prepared and used as the source of the deaminase activity. 

In the study by Raffin and Thebault (1991), AMP and IMP were separated on a Partisphere SAX column (4.6 mm x 125 mm, 5/u.m). The mobile phase was composed of 40 mM potassium phosphate buffer (pH 3.5) and the flow rate was 1 mL/min. The effluent was monitored at 254 nm and peaks were quantified using peak heights and standard solutions of AMP and IMP. 

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DiaZepin Nucleosides. Four naturally occurring dia2epin nucleosides, coformycin (58), 2 -deoxycoformycin (59), adechlorin or 2 -chloro-2 -deoxycoformycin (60), and adecypenol (61), have been isolated (1—4,174,175). The biosynthesis of (59) and (60) have been reported to proceed from adenosine and C-1 of D-ribose (30,176,177). They are strong inhibitors of adenosine deaminase and AMP deaminase (178). Compound (58) protects adenosine and formycin (12) from deamination by adenosine deaminase. Advanced hairy cell leukemia has shown rapid response to (59) with or without a-or P-interferon treatment (179—187). In addition, (59) affects interleukin-2 production, receptor expression on human T-ceUs, DNA repair synthesis, immunosuppression, natural killer cell activity, and cytokine production (188—194). 

The lower panel shows the decreasing concentration of ATP, to about 60% of resting levels, and the simultaneous equimolar increase in IMP. The fall in ATP started when most of the PCr store was utilized, resulting in a decreased rate of ADP phosphorylation via the creatine kinase reaction. The resultant accumulation of ADP stimulates adenylate kinase activity and subsequently IMP is formed via the AMP deaminase reaction ... 

Attention has been drawn to the potential of phosphoric acid anhydrides of nucleoside 5 -carboxylic acids (14) as specific reagents for investigating the binding sites of enzymes. For example, (14 B = adenosine) inactivates adenylosuccinate lyase from E. coli almost completely, but has little effect on rabbit muscle AMP deaminase. The rate of hydrolysis of (14) is considerably faster than that of acetyl phosphate, suggesting intramolecular assistance by the 3 -hydroxyl group or the 3-nitrogen atom. 

JMC3596>, an imidazo[4,5-d][l,3]diazepin-8-ol as an AMP deaminase inhibitor <00JMC1508>, the antimaiarial mode of action of artemisinin <00FEBSL238>, and finally dithiepin-l,l,4,4-tetroxides as non-peptidic human galanin receptor antagonists <00BMC1383>. 

Gruber, and J. R. Appleman, Discovery of AMP mimetics that exhibit high inhibitory potency and specificity for AMP deaminase, J. Am. Chem. Soc. 121 308 (1999). 

Deficiency of the muscle-specific myoadenylate deaminase (MADA) is a frequent cause of exercise-related myopathy and is thought to be the most common cause of metabolic myopathy. MADA catalyzes the deamination of AMP to IMP in skeletal muscle and is critical in the purine nucleotide cycle. It is estimated that about 1-2% of all muscle biopsies submitted to medical centers for pathologic examination are deficient in AMP deaminase enzyme activity. MADA is 10 times higher in skeletal muscle than in any other tissue. Increase in plasma ammonia (relative to lactate) after ischemic exercise of the forearm may be low in this disorder, which is a useful clinical diagnostic test in patients with exercise-induced myalgia... 

Genetta, T., Morisaki, H., Morisaki, T. and Holmes, E. W. A novel bipartite intronic splicing enhancer promotes the inclusion of a mini-exon in the AMP deaminase 1 gene. J. Biol. Chem. 276 25589-25597,2001. 

The observed normal isotope effect of 1.9 provides further evidence supporting the role of Asp55 as the general base. Namely, a normal isotope effect of 1.9 is most consistent with general base catalysis by an amino acid side chain, as inverse isotope effects are commonly observed when a zinc-bound water molecule, or hydroxide, is the attacking nucleophile. For example, the zinc-containing enzymes AMP deaminase [111], thermolysin [112], stromelysin [113], and a desuccinylase [114] are each believed to utilize a zinc-bound water as the nucleophile, and all of these reactions are characterized by an inverse deuterium isotope effect. This inverse isotope effect is thought to result from a dominant... 

The AK reaction could lead to significant accumulation of AMP. Ideally rephosphorylation of AMP to ATP would occur to maintain ATP concentration but this is not really an option because the bioenergetics of such a reaction are so unfavourable, therefore an alternative mechanism of recycling AMP is required. An enzyme called AMP deaminase (AD) is important in this process. The activity of AMP deaminase is really one of AMP removal rather than recycling of AMP to ATP. 

The enzyme AMP deaminase is inactive in the sarcoplasm in a resting muscle but is activated by (i) low pH and (ii) a reduction in the [ATP] -to- [AMP] ratio both of these changes are consistent with actively working skeletal muscle. 

MTX also has several effects on the purine synthetic pathway. MTXPGs inhibit the enzyme aminoimidazole carboxamide ribonucleotide (AlCAR) transformylase, which in turn causes intracellular accumulation of AICAR. AICAR and its metabolites can then inhibit two enzymes in the adenosine pathway adenosine deaminase and adenosine monophosphate (AMP) deaminase, which leads to intracellnlar accumulation of adenosine and adenine nucleotides. Subsequent dephosphorylation of these nucleotides results in increased extracellular concentrations of adenosine, which is a powerful anti-inflammatory agent (11). 

Within a cell, a nncleotidase catalyses the hydrolysis of either a ribonncleotide or deoxyribonucleotide (Fignre 10.8). The qnantitatively important pathway for degradation of AMP in liver and mnscle involves deamination to IMP, catalysed by AMP deaminase, producing ammonia, and snbseqnent hydrolysis of IMP to inosine. This may be an important sonrce of inosine for synthesis of phosphati-dylinositol, a key phospholipid in membranes. 

Vasodilation is stimulated by an increase in the concentration of adenosine. The enzyme AMP deaminase catalyses the formation of adenosine from AMP as follows ... 

Figure 22.17 Summary of mechanisms to maintain the ATP/ADP concentration ratio in hypoxic myocardium. A decrease in the ATP/ADP concentration ratio increases the concentrations of AMP and phosphate, which stimulate conversion of glycogen/ glucose to lactic acid and hence ATP generation from glycolysis. The changes also increase the activity of AMP deaminase, which increases the formation and hence the concentration of adenosine. The latter has two major effects, (i) It relaxes smooth muscle in the arterioles, which results in vasodilation that provides more oxygen for aerobic ATP generation (oxidative phosphorylation). (ii) It results in decreased work by the heart (i.e. decrease in contractile activity), (mechanisms given in the text) which decreases ATP utilisation.

By reactions of different 13,4-triazine derivatives, C-ribosyl imidazo[2,l-/][13,4]triazines <99JCS(P1)2929> and C-ribosyl 13.4-triazolo[3,4-/ [13,4]triazines <99JCS(P1)2937> have been synthesized as inhibitors of adenosine and AMP deaminases. Catalytic asymmetric aminohydroxylation with amino substituted 13,4-triazine and 133-tiiazine derivatives, as nitrogen sources, has been described <99AG(E)1080>. 

Muscular work is accompanied by the production of ammonia, the immediate source of which is adenosine 5 -phosphate (AMP).301 302 This fact led to the recognition of another substrate cycle (Chapter 11) that functions by virtue of the presence of a biosynthetic pathway and of a degradative enzyme in the same cells (cycle A, Fig. 25-17). This purine nucleotide cycle operates in the brain303 304 as well as in muscle. The key enzyme 5-AMP aminohydrolase (AMP deaminase step a, Fig. 25-17) also occurs in erythrocytes and many other tissues.304 305 Persons having normal erythrocyte levels but an absence of this enzyme in muscles suffer from muscular weakness and cramping after exercise.306... 

The enzyme from M. audouini cultured on wheat bran, initially called AMP deaminase (185), has been extensively purified (4000-fold) (188) and renamed ATP aminohydrolase because of greater activity on that substrate. The ultracentrifugal sedimentation pattern exhibited a large and small peak, but no evidence was presented regarding the identity of either. Requirements for catalytic cofactors were negative. 

Carbon-14 and nitrogen-15 heavy-atom KIEs in the hydrolytic deamination of adenosine 5-monophoshate (AMP 513) with AMP deaminase... 

SCHEME 4.12 Coformycin (4.11), a transition state inhibitor of AMP deaminase... 

M. Yoshino and K. Murakami (1988). A kinetic study of the inhibition of yeast AMP deaminase by polyphosphate. Biochim. Biophys. Acta, 954, 271-276. 

Myoadenylate deaminase deficiency is a recessive disorder that affects approximately 1 -2% of populations of European descent, but appears considerably rarer in Asian populations. Myoadenylate deaminase, also called adenosine monophosphate (AMP) deaminase, is an enzyme that converts AMP to inosine monophosphate (IMP). Its deficiency results in excess AMP, which is lost by excretion with disturbances in energy generation. Symptoms of severe fatigue and muscle pain can result. 

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