NAD+-dependent 15-hgydroxyprostaglandin dehydrogenase from porcine kidney. II. Kinetic studies.

The kinetic mechanism of porcine renal NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (11 alpha, 15-dihydroxy-9-oxoprost-13-enoate:NAD+ 15-oxidoreductase, EC 1.1.1.141) was investigated. Initial velocity studies gave intersecting double reciprocal plots that conform to a sequential mechanism. Product inhibition studies indicated that 15-keto-prostaglandin E2 exhibited linear non-competitive inhibtion with respect to either prostaglandin E2 or NAD+, and NADH yielded linear competitive inhibition with respect to NAD+. Dead-end inhibition studies showed that adenosine-5'-diphosphoribose inhibited the enzyme competitively with respect to NAD+ as expected, but inhibited the enzyme non-competitively with respect to prostaglandin, E2. Alternate substrate studies indicated that a mixture of 3-acetyl-NAD+ and NAD+ gave a concave upward double reciprocal plot, while a mixture of prostaglandin E2 and prostaglandin F2 alpha yielded a linear plot. These results are consistent with an ordered Bi-Bi mechanism where NAD+ is added first, followed by prostaglandin E2, and 15-keto-prostaglandin E2 is released, followed by NADH.