title: Short-chain dehydrogenases. Proteolysis and chemical modification of prokaryotic 3 alpha/20 beta-hydroxysteroid, insect alcohol and human 15-hydroxyprostaglandin dehydrogenases.
abstract: Prokaryotic 3 alpha/20 beta-hydroxysteroid dehydrogenase exhibits one segment sensitive to proteolysis with Glu-C protease and trypsin (cleaving after Glu192 and Arg196, respectively). Cleavage is associated with dehydrogenase inactivation; the presence of NADH offers almost complete protection and substrate (cortisone) gives some protection. Distantly related insect alcohol dehydrogenase is more resistant to proteolysis, but cleavage in a corresponding segment is detectable with Asp-N protease (cleaving before Asp198), while a second site (at Glu243) is sensitive to cleavage with both Glu-C and Asp-N proteases. Combined, the results suggest the presence of limited regions especially sensitive to proteolysis and the possibility of some association between the enzyme active site and the sensitive site(s). Modification of the hydroxysteroid dehydrogenase with tetranitromethane is paralleled by enzyme inactivation. With a 10-fold excess of reagent, labeling corresponds to 1.2 nmol Tyr/nmol protein chain and is recovered largely in Tyr152, with lesser amounts in Tyr251. Tetranitromethane also rapidly inhibits the other two dehydrogenases, but they contain Cys residues, preventing direct correlation with Tyr modification. Together, the proteolysis and chemical modifications highlight three segments of short-chain dehydrogenase subunits, one mid-chain, containing Tyr152 of the steroid dehydrogenase (similar numbers in the other enzymes), strictly conserved and apparently close to the enzyme active site, the other around position 195, sensitive to proteolysis and affected by coenzyme binding, while the third is close to the C-terminus.
authors.affiliation: Department of Chemistry I, Karolinska Institutet, Sweden.
authors.forename: M
authors.initials: M
authors.lastname: Krook
authors.forename: V
authors.initials: V
authors.lastname: Prozorovski
authors.forename: S
authors.initials: S
authors.lastname: Atrian
authors.forename: R
authors.initials: R
authors.lastname: Gonzàlez-Duarte
authors.forename: H
authors.initials: H
authors.lastname: Jörnvall
mesh_terms: D000426:Alcohol Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003349:Cortisone Reductase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism*; D006801:Humans; D006911:Hydroxyprostaglandin Dehydrogenases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D009243:NAD / Q000494:pharmacology; D012697:Serine Endopeptidases / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology; D013774:Tetranitromethane / Q000494:pharmacology; D014357:Trypsin / Q000378:metabolism
chemical_list: D009243:NAD; D006911:Hydroxyprostaglandin Dehydrogenases; D000426:Alcohol Dehydrogenase; C035859:15-hydroxyprostaglandin dehydrogenase; D003349:Cortisone Reductase; D010450:Endopeptidases; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin; D013774:Tetranitromethane