INOSINE 5'-PHOSPHATE DEHYDROGENASE . KINETIC MECHANISM AND EVIDENCE FOR SELECTIVE REACTION OF 6-CHLORO ANALOG OF INOSINE 5'-PHOSPHATE WITH A CYSTEINE RESIDUE AT INOSINE 5'-PHOSPHATE SITE

A purified preparation of inosine 5′-phosphate dehydrogenase of Aerobacter aerogenes showed with polyacrylamide disc electrophoresis two major protein components and two minor components which possessed the dehydrogenase activity and several trace protein components which lacked that activity. Ultracentrifugation of the preparation showed two peaks with sedimentation coefficients (s20,w) of 9.1 and 12.1 S, respectively. Product inhibition and initial velocity data were consistent with one kinetic mechanism involving two substrates and with two mechanisms which include as a third substrate the source (presumably water) of the oxygen at carbon atom 2 of xanthosine 5′-phosphate. All three of these possible mechanisms are ordered, with inosine 5′-phosphate the first substrate to bind and xanthosine 5′-phosphate the last product to leave. The inhibitor constant of xanthosine 5′-phosphate was 1.2 X 10-4 M at pH 8.1 and 24°. The rate of inactivation of inosine 5′-phosphate dehydrogenase by different levels of 6-chloropurine ribonucleoside 5′-phosphate followed saturation kinetics. The apparent dissociation constant of the 6-chloropurine nucleotide-enzyme complex at pH 7.0 and 24° was 2.6 × 10-4 M and the apparent first-order rate constant for inactivation was 0.125 sec-1. Titration of the enzyme with inhibitor showed that inactivation was paralleled by the disappearance of the absorption maximum at 263 mμ of 6-chloropurine nucleotide and the appearance of a maximum at 292 mμ which in wavelength and intensity corresponded to a 6-alkyl-mercaptopurine nucleotide. Inosine 5′-phosphate slowed the rate of spectral change and the rate of inactivation in equal proportion. High levels of 6-chloropurine ribonucleoside caused no enzyme inactivation or spectral changes, presumably because this nucleoside, like inosine, has no affinity for the inosine 5′-phosphate site of the dehydrogenase. At pH 9 and 24° 6-chloropurine nucleoside reacted with mercaptide ion five to eight times faster than did its 5′-phosphate derivative. It is concluded that the 6-chloropurine nucleotide exclusively derivatizes cysteine residues located at the inosine 5′-phosphate sites of the dehydrogenase and that approximately 2 molar equiv inactivate the enzyme if it is assigned a molecular weight of 200,000 from the sedimentation coefficient of 9.1 S. © 1968, American Chemical Society. All rights reserved.