ANALOGS OF INOSINE 5'-PHOSPHATE WITH PHOSPHORUS - NITROGEN AND PHOSPHORUS - SULFUR BONDS . BINDING AND KINETIC STUDIES WITH INOSINE 5'-PHOSPHATE DEHYDROGENASE

Inosine 5′-phosphorothioate, 5′-mercapto-5′-deoxyinosine 5′-N-phosphate, and 5′-amino-5′-deoxyinosine 5′- A-phosphate have been synthesized and found to be substrates of inosine 5′-phosphate dehydrogenase of Aerobacter aerogenes. Initial velocity data at the pH optimum (8.1) was consistent with an ordered kinetic model, presumably of the BiBi type which is consistent with kinetic data when inosine 5′- phosphate is substrate, and in which inosine 5′-phosphate is the first substrate added. On this basis, enzyme-substrate dissociation constants of 23 × 10-5, 1.7 × 10-5, and 0.5 × 10-5 m, respectively, were determined for the above nucleotide analogs (cf inosine 5′-phosphate, 2.0 × 10-5 m). The Michaelis constants were 21 × 10-5, 1.3 × 10-5, and 3.8 × 10-5 m, and the Vmhx values relative to that for inosine 5′-phosphate (Lma, = 1) were 1.05, 0.75, and 0.67, respectively. The kinetic properties of inosine 5′-phosphorothioate can be ascribed entirely to a tenfold reduction in the rate constant for interaction of substrate and enzyme which accompanies this modification of the inosine 5′-phosphate structure. With the 5′-S-phosphate analog, the rate constants for enzyme-substrate association or dissociation were the same as those of inosine 5′- phosphate; with the 5′-A-phosphate. a reduction in both rate constants is observed. The C-5′ oxygen of inosine 5′-phosphate is concluded not to contribute significantly to the total binding energy. The present studies appear to favor the view that inosine 5′-phosphate binds preferentially in its phosphodianion form. Ultraviolet optical rotatory dispersion determinations in aqueous solution provided no evidence for major conformational differences between the analogs and inosine 5′- phosphate itself. At pH 6 the kinetic mechanism was the same as at pH 8.1; changes in the rate constants for enzyme-substrate interaction of inosine 5′-phosphate and its 5′-A-phosphate analog at this pH are ascribed to changes in the enzyme. © 1969, American Chemical Society. All rights reserved.