Properties of 5'-AMP deaminase and its inhibitors with the aid of a continuous fluorimetric assay with formycin-5'-phosphate as substrate.
A new continuous fluorimetric assay for AMP deaminase activity is described. The method makes use of a fluorescent analog of 5'-AMP, formycin-5'-phosphate (5'-FMP), which undergoes deamination to formycin B-5'-phosphate, not fluorescent at neutral pH. The pH-dependence for deamination of 5'-FMP is similar to that for 5'-AMP, but shifted about 0.2 units to more acidic pH. Deamination of 5'-FMP may also be followed spectrophotometrically at 306 nm, permitting better assays of crude extracts. Some kinetic results obtained by means of the new method for AMP deaminase from chick and rabbit skeletal muscle are presented. In particular it was found that the natural product of deamination, 5'-IMP exhibited allosteric inhibition of the chick enzyme with Ki values 1.6 mM, 1.2 mM and 1.0 mM at pH 5.8, 6.5 and 7.3, respectively. Activation by diadenosine tetraphosphate, Ap4A, reported for mouse muscle AMP deaminase, has not been noted for the chick enzyme. Inhibition by the transition state analogs, coformycin and 2'-deoxycoformycin, was observed for both rabbit and chick deaminases with Ki values approximately 1 microM and approximately 1.6 microM respectively. Kinetic data for coformycin-5'-phosphate show it to be a tight-binding inhibitor with Ki less than 0.6 x 10(-9) M as compared to 1 x 10(-9) M for 2'-deoxycoformycin-5'-phosphate.
Bzowska
A
A
Department of Biophysics, University of Warsaw, Poland.
Shugar
D
D
D000659:AMP Deaminase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000249:Adenosine Monophosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D000903:Antibiotics, Antineoplastic; D002645:Chickens; D005573:Formycins; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D009709:Nucleotide Deaminases / Q000378:metabolism*; D011817:Rabbits; D012265:Ribonucleotides; D013050:Spectrometry, Fluorescence / Q000379:methods; D013379:Substrate Specificity
D000903:Antibiotics, Antineoplastic; D005573:Formycins; D012265:Ribonucleotides; C021683:formycin 5'-phosphate; D000249:Adenosine Monophosphate; D009709:Nucleotide Deaminases; D000659:AMP Deaminase