AMP aminohydrolase

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... 

Inhibition of the chicken breast enzyme by rabbit antisera for chicken breast enzyme the lack of effect on the chicken brain, heart, or erythrocyte enzyme and the differences in substrate specificity exhibited by the brain and breast muscle enzyme are consistent with at least two isozymes of chicken 5 -AMP aminohydrolase (123). Isozymic patterns, while perhaps implied by differences in certain kinetic pa-... 

Treatment of 9-(/ -D-ribofuranosyluronic acid)adenine with diphenylphosphoro-chloridate and orthophosphate or tripolyphosphate yields (62) and (63), which, although unstable, inhibit rabbit AMP aminohydrolase and pyruvate kinase, respectively, with behaviour characteristic of active-site-specific reagents.98 Adenylate kinases from several sources are inactivated by iV6-[2- and 4-fluorobenzoyl]-adenosine-5 -triphosphates, with kinetics characteristic of active-site labelling, although these compounds were without effect on yeast hexokinase and rabbit pyruvate kinase.99... 

The existence of two separate enzymes in animal tissues responsible for the liberation of ammonia from each of the two aminopurines, adenine and guanine, the latter specific for the free purine and the former for the nucleosides, was initially presented by Jones and his colleagues 11, 12). In 1928, Schmidt 13-15) demonstrated that AMP aminohy-drolase was responsible for the appearance of inosinic acid in muscle and for at least a portion of ammonia liberated during contraction. He showed not only a marked specificity for deamination of 5 -AMP but also provided the first clue that muscle adenylic acid (5 -AMP) and yeast adenylic acid (3 -AMP) were different compounds. Initial evidence for guanine and adenosine aminohydrolase including aspects of the specificity were also described by Schmidt 16). Additional details regarding development of interest in purine aminohydrolases are available in several excellent reviews 17-20). 

In general, AMP aminohydrolase specificities have not been thoroughly defined perhaps because of difficulties until recently in obtaining pure enzyme. In addition to AMP and dAMP, the muscle enzyme catalyzes the deamination of V -methyl AMP, iV -ethyl AMP, for-mycin-5 -monophosphate, adenosine-5 -monosulfate, adenosine-5 -phos-phoramidate, adenosine, ADP (133), adenosine-5 -phosphorothioate, and 6-chloropurine 5 -ribonucleotide (124) ATP, GMP, CMP, 2 -AMP, 3 -AMP, 3, 5 -cyclic AMP, 3-iso-AMP, V-methyl AMP, toyocamycin-5 -monophosphate, tubercidin-5 -monophosphate, and 6-mercaptopurine-5 -ribonucleotide are not deaminated (133). The elasmobranch fish muscle, carp muscle, and avian brain enzymes appear to be specific for AMP and dAMP (123, 125, 126). Extracts from pea seed and erythrocytes and the purified calf brain enzyme are specific for AMP (48, 131, 134). 

The kinetic parameters of various muscle AMP aminohydrolases presented in Table V (51, 68, 122, 124-127) are similar except for the lower specific activities exhibited by the fish enzymes for which no criteria of homogeneity are presently available. Specific activities reported for brain enzymes not shown in Table V are 15 /amoles/min/mg for calf (129) and 30 /amoles/min/mg for chicken (123). Although the pH optimum for AMP deamination varies depending upon the source, it normally occurs in a range from pH 5.9 to 7.1 (48, 125, 126, 129, 130, 135-137). 

AMP aminohydrolase from rabbit muscle. All were competitive inhibitors for the enzyme, and many were substrates however, the a-L-/a/o-epimer of 5 -C-propionyl-aminomethyl-AMP was found to be a non-competitive inhibitor, though it showed no... 

AMP deaminase reductase (deaminating) (pyridoxal containing) AMP aminohydrolase 3.5.4.6 14... 

The acyl phosphate (I) hydrolyzes in Tris buffer at pH 7.7 at least 100-fold faster than does acetyl phosphate, as shown by the inability of (I) to inactivate adenylosuccinate-lyase after a 15-sec exposure to the buffer before addition of the enzyme. Compound (I) also lost its ability to inactivate AMP aminohydrolase after 15-sec hydrolysis at pH 6.5. After the solution of (I) in iV,A -dimethylformamide had been stored at —25° for 2 days, it showed UV spectral changes (upon dilution into aqueous solutions) indicative of A -acylaminopurine nucleoside formation. In the case of AMP aminohydrolase, this change is associated with a marked reduction in the degree of enzyme inactivation, and the use of freshly prepared solutions of (I) in studies of enzyme inactivation is therefore indicated. 

Initial reaction velocities were measured at 340 nm. For all experiments (except where noted) the final volume of 1.00 ml of 0.1 M Tris chloride (pH 7.6) contained 13 /ug of rabbit muscle lactic dehydrogenase, 0.05 jug of rabbit muscle pyruvate kinase, 0.1 M KCl, 0.025 M MgS04, 1.5 mM phosphoenolpyruvate (sodium salt), 0.25 mAf ADP (sodium salt), and 0.25 mM NADH. The order of addition of the components was the same as described above for AMP aminohydrolase. 

After purine nucleotides have been converted to the corresponding nucleosides by 5 -nucleotidases and by phosphatases, inosine and guanosine are readily cleaved to the nucleobase and ribose-1-phosphate by the widely distributed purine nucleoside phosphorylase. The corresponding deoxynucleosides yield deoxyribose- 1-phosphate and base with the phosphorylase from most sources. Adenosine and deoxyadenosine are not attacked by the phosphorylase of mammalian tissue, but much AMP is converted to IMP by an aminohydrolase (deaminase), which is very active in muscle and other tissues (fig. 23.20). An inherited deficiency of purine nucleoside phosphorylase is associated with a deficiency in the cellular type of immunity. 

Purine nucleotide catabolism is outlined in Figure 15.12. There is some variation in the specific pathways used by different organisms or tissues to degrade AMP. In muscle, for example, AMP is initially converted to IMP by AMP deaminase (also referred to as adenylate aminohydrolase). IMP is subsequently hydrolyzed to inosine by 5 -nucleotidase. In most tissues, however, AMP is hydrolyzed by 5 -nucleotidase to form adenosine. Adenosine is then deaminated by adenosine deaminase (also called adenosine aminohydrolase) to form inosine. 

Direct deamination of AMP to IMP by adenylate aminohydrolase (EC 3.5.4.6) occurs primarily in mammalian systems. It plays little, if any, role in bacteria where conversion usually occurs indirectly via deamination of adenine or adenosine [113,114]. Little is known about regulation at these levels, although adenosine deaminase is inducible by its substrate [114] and mutants lacking it have been obtained [115], Another indirect conversion in bacteria involves the regeneration of AlCAR, in the eventual conversion of phosphoribosyl-ATP (PR-ATP) to histidine. The AlCAR so obtained reenters the biosynthetic pathway and IMP is produced [33]. This pathway is regulated by histidine which exerts a profound feedback inhibition at the level of PR-ATP formation from ATP [116]. 

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