title: Transition-metal complexes as enzyme-like reagents for protein cleavage: complex cis-[Pt(en)(H2O)2]2+ as a new methionine-specific protease.
abstract: Complex cis-[Pt(en)(H(2)O)(2)](2+) promotes selective hydrolytic cleavage of two proteins, horse cytochrome c and bovine beta-casein. The cleavage is completed in 24 h under relatively mild conditions, at about pH 2.5, and a temperature as low as 40 degrees C. The results of HPLC and TSDS PAGE separations, MALDI mass spectrometry, and Edman sequencing showed that cleavage occurred exclusively at the peptide bond involving the C-terminus of each methionine residue, both such residues in cytochrome c and all six such residues in beta-casein. While having the same selectivity as cyanogen bromide (CNBr), the most common chemical protease, cis-[Pt(en)(H(2)O)(2)](2+) has several advantages. It is nonvolatile, easy to handle, and recyclable. Its cleavage is residue-selective, the rest of the polypeptide backbone remains intact, and the other side chains remain unmodified. It is applied in approximately equimolar amounts with respect to methionine residues, creates free amino and carboxylic groups, and cleaves even the Met-Pro bond, which is resistant to CNBr and most proteolytic enzymes. Finally the complex also works in the presence of the denaturing reagent sodium dodecyl sulfate. Experiments with the synthetic peptides, AcAla-Lys-Tyr-Gly-Gly-Met-Ala-Ala-Arg-Ala (termed Met-peptide) and AcVal-Lys-Gly-Gly-His-Ala-Lys-Tyr-Gly-Gly-Met-Ala-Ala-Arg-Ala (termed HisMet-peptide) as substrates, revealed structural and mechanistic features of the proteolytic reactions. We explain why two similar complexes with similar metal ions, cis-[Pt(en)(H(2)O)(2)](2+) and cis-[Pd(en)(H(2)O)(2)](2+), differ in selectivity as proteolytic reagents. The selectivity of cleavage is governed by the selectivity of the cis-[Pt(en)(H(2)O)(2)](2+) binding to the methionine side chain. The proteolytic activity is governed by the modes of coordination, which control the approach of the anchored Pt(II) ion to the scissile peptide bond. The cleavage occurs with a small, but significant, catalytic turnover of more than 18 after 7 days. The ability of cis-[Pt(en)(H(2)O)(2)](2+) to cleave proteins at relatively few sites, with explicable selectivity and catalytic turnover, bodes well for its use in biochemical practice.
authors.affiliation: Department of Chemistry, Gilman Hall, Iowa State University, Ames, IA 50011-3111, USA.
authors.forename: Nebojsa M
authors.initials: NM
authors.lastname: Milović
authors.forename: Laura-M
authors.initials: LM
authors.lastname: Dutca
authors.forename: Nenad M
authors.initials: NM
authors.lastname: Kostić
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002364:Caseins / Q000378:metabolism; D002417:Cattle; D003488:Cyanogen Bromide / Q000737:chemistry / Q000378:metabolism; D045304:Cytochromes c / Q000378:metabolism; D010450:Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D006736:Horses; D007202:Indicators and Reagents; D007700:Kinetics; D008715:Methionine / Q000737:chemistry / Q000378:metabolism*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D015394:Molecular Structure; D008970:Molecular Weight; D010165:Palladium / Q000737:chemistry / Q000378:metabolism; D017671:Platinum Compounds / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D002364:Caseins; D007202:Indicators and Reagents; D017671:Platinum Compounds; D011506:Proteins; D010165:Palladium; D045304:Cytochromes c; D008715:Methionine; D010450:Endopeptidases; D003488:Cyanogen Bromide