Dissociation and reconstitution of bovine seminal RNAase: construction of a hyperactive hybrid dimer.
The quaternary structure of bovine seminal ribonuclease, the only dimeric protein in the superfamily of ribonucleases, is maintained both by noncovalent forces and by two intersubunit disulfides. The available monomeric derivatives of the enzyme may not be reassembled into dimers. They are catalytically active, but do not retain certain properties of the dimeric enzyme, such as: (i) the ability to respond cooperatively to increasing substrate concentrations in the rate-limiting reaction step; and (ii) the antitumor and immunosuppressive actions. In this report we described the preparation of stable monomers of seminal ribonuclease which can be reassociated into covalent dimers indistinguishable from the native protein. With this procedure a hybrid dimer was constructed, made up of a native subunit associated to a subunit catalytically inactivated by selective alkylation of the active site His-119. This dimer was found to have enzymic properties typical of monomeric ribonucleases, such as a hyperbolic saturation curve in the hydrolytic rate-limiting step of the reaction. However, the hybrid dimer was one order-of-magnitude more active than the dimeric enzyme.
Tamburrini
M
M
Department of Organic and Biological Chemistry, University of Naples.
Piccoli
R
R
Picone
D
D
Di Donato
A
A
D'Alessio
G
G
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D004220:Disulfides / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D008297:Male; D008970:Molecular Weight; D010084:Oxidation-Reduction; D055503:Protein Multimerization; D012260:Ribonucleases / Q000378:metabolism*; D012661:Semen / Q000201:enzymology*; D012669:Seminal Vesicles / Q000201:enzymology*; D013438:Sulfhydryl Compounds / Q000378:metabolism
D000596:Amino Acids; D004220:Disulfides; D046911:Macromolecular Substances; D013438:Sulfhydryl Compounds; D012260:Ribonucleases; D004229:Dithiothreitol