CYSTEINE-182 IS ESSENTIAL FOR ENZYMATIC-ACTIVITY OF HUMAN PLACENTAL NAD(-DEPENDENT 15-HYDROXYPROSTAGLANDIN DEHYDROGENASE())

Evidence suggests that one or more cysteine residues may be important for the activity of human placental NAD(+)-dependent 15-hydroxyprostag landin dehydrogenase (15-PGDH), All of these four cysteines (Cys 45, C ys 63, Cys 152, and Cys 182) are found in areas which are believed to be important for the functioning of the enzyme, Site-directed mutagene sis was used to examine the role of the four cysteine residues found i n 15-PGDH, Each cysteine was individually changed to an alanine and to phenylalanine, The C182A mutant protein was completely inactive, whil e the other three alanine mutants retained full activity, When all of the cysteines were individually changed to phenylalanine, only the C45 F mutant retained full activity, The C63F mutant enzyme retained only about 10% of the wild-type activity while the C152F and C182F mutants were inactive, From these results it appears that only C182 is necessa ry for enzyme activity, Mutagenesis of Cys 63 and Cys 152 to phenylala nine lends support to the suggestion that these two residues are locat ed in critical parts of the enzyme. (C) 1996 Academic Press, Inc.