Thiol protease-like active site found in the enzyme dienelactone hydrolase: localization using biochemical, genetic, and structural tools.
The active site of dienelactone hydrolase (DLH), a microbial enzyme of the beta-ketoadipate pathway, has been conclusively located using a combination of crystallographic, biochemical, and genetic techniques. DLH hydrolyzes a dienelactone to maleylacetate and has esterase activity on p-nitrophenyl acetate and trans-cinnamoyl imidazole. The identification of Cys-123 as containing the essential thiol confirms the localization of the active site as suggested by the crystal structure of DLH, and disproves an earlier hypothesis regarding its location. Two mutant proteins have been engineered in which Cys-123 has been converted to a serine (C123S DLH) and an alanine (C123A DLH), respectively. C123S DLH (Km = 9900 +/- 2300 microM; Vmax = 4.4 +/- 0.8 mumol/min-mg) displays burst kinetics with p-nitrophenyl acetate and is 10% as active as DLH (Km = 170 +/- 7 microM; Vmax = 21.1 +/- 0.4 mumol/min-mg). C123A DLH is inactive. The structures of DLH, C123S DLH, and C123A DLH have been refined at 1.8, 2.2, and 2.0 A, respectively. Comparison of the structures of these proteins demonstrates that the only differences between them are centered at residue 123. The structures of the active sites of DLH, papain, and subtilisin are similar and are suggestive of the three enzymes having evolved convergently to similar active sites with similar enzymic mechanisms.
Pathak
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Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois 60208.
Ashley
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Ollis
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D001483:Base Sequence; D001665:Binding Sites; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000235:genetics; D003196:Computer Graphics; D005583:Fourier Analysis; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010206:Papain / Q000737:chemistry; D011487:Protein Conformation; D013237:Stereoisomerism; D013381:Subtilisins / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D014961:X-Ray Diffraction
D013438:Sulfhydryl Compounds; D002265:Carboxylic Ester Hydrolases; C046130:carboxymethylenebutenolidase; D013381:Subtilisins; D010206:Papain