Threonine 188 is critical for interaction with NAD(+) in human NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase

NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is the key enzyme in the inactivation pathway of prostaglandins, It is a member of th e short-chain dehydrogenase family of enzymes. A relatively conserved threo nine residue corresponding to threonine 188 of 15-PGDH is proposed to be in volved in the interaction with the carboxamide group of NAD(+), Site-direct ed mutagenesis was used to examine the important role of this residue. Thre onine 188 was changed to alanine (T188A), serine (T188S) or tyrosine (T188Y ) and the mutant proteins were expressed in E. coli. Western blot analysis showed that the expression levels of mutant proteins were similar to that o f the wild type protein. Mutants T188A and T188Y were found to be inactive. Mutant T188S still retained substantial activity and the Km value for PGE, was similar to the wild enzyme; however, the Km value for NAD(+) was incre ased over 100 fold. These results suggest that threonine 188 is critical fo r interaction with NAD(+) and contributes to the full catalytic activity of 15-PGDH, (C) 1999 Academic Press.