Properties of 5′-AMP Deaminase and its Inhibitors with the Aid of a Continuous Fluorimetric Assay with Formycin-5′-phosphate as Substrate

Abstract

A new continuous fluorimetric assay for AMP deaminase activity is described. The m ethod makes use of a fluorescent analog of 5′-AMP, formycin-5′-phosphate (5′-FM P), which undergoes deam ination 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′-FM P 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 K, values 1.6mM , 1.2mM and 1.0mM 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 ~ 1 μM and ~ 1.6 μM respectively. Kinetic data for coformycin-5′-phosphate show it to be a tight-binding inhibitor with K, < 0.6 × 10-9 M as compared to 1 × 10-9 m for 2′-deoxycoformycin-5′-phosphate.