C-Terminal region of human NAD+-dependent 15-hydroxyprostaglandin dehydrogenase is involved in the interaction with prostaglandin substrates.
NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) catalyzes the oxidation of the 15(S) hydroxyl group of prostaglandins to a 15-keto group resulting in a significant reduction of the biological activities of prostaglandins. Although the key residues involved in NAD+ binding and in catalytic activity have been partially identified, the sites of interaction of the enzyme with the prostaglandin substrates are yet to be determined. Homology analysis of the primary structures of 15-PGDH from human, mouse and rat indicates that the sequences are almost homologous except for two regions near the C-terminus. The involvement of the C-terminal region in catalytic activity was examined by studies on C-terminally truncated enzymes and on human/rat chimeric enzymes. When three to four amino acids were removed successively from the C-terminal end of human 15-PGDH, the truncated enzymes exhibited decreasing Vmax/Km ratios and increasing Km values for PGE2 as the chain was shortened. Similarly, when the C-terminal 14 amino acids of human 15-PGDH were replaced by the C-terminal 14 amino acids of rat 15-PGDH or vice versa, the Vmax/Km ratios and the Km values for prostaglandin E2 of the chimeric enzymes were in between those of the two wild-type enzymes. This indicates that the catalytic effectiveness of human 15-PGDH decreases as the C-terminal region is gradually removed or replaced by rat sequences. The C-terminal region appears to be more important for the interaction of the enzyme with the prostaglandin substrates than with the coenzyme.
Zhou
H
H
Division of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY 40536-0082, USA.
Yan
F
F
Tai
H H
HH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002384:Catalysis; D017931:DNA Primers; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006911:Hydroxyprostaglandin Dehydrogenases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism*; D011453:Prostaglandins / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013379:Substrate Specificity
D017931:DNA Primers; D018076:DNA, Complementary; D011453:Prostaglandins; D011993:Recombinant Fusion Proteins; D009243:NAD; D006911:Hydroxyprostaglandin Dehydrogenases; C035859:15-hydroxyprostaglandin dehydrogenase