Metal Complex Bioconjugates as Artificial Enzymes

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This award in the Inorganic, Bioinorganic and Organometallic Chemistry program supports research by Dr. Nenad M. Kostic at Iowa State University to synthesize reagents that catalytically cleave proteins with unprecedented sequence-selectivity and site-specificity. These inorganic reagents complement enzymes in applications in biochemistry, molecular biology, pharmaceutical chemistry, and biotechnology. This research builds on previous NSF-supported work that explained a striking difference in the sequence-selectivity of Pd(II) and Pt(II) complexes as cleavage agents in terms of the coordination stereochemistry and the kinetics and mechanisms of the cleavage reactions. The current research aims to develop a new biotechnological method for protein cleavage by Pd complexes and also to develop a second generation of Pd-containing cleavage reagents that will achieve catalytic turnover in backbone cleavage. The specific objectives are to: (1) study the effects of the residue X in the X-Pro and X-Pro-His segments and of the local protein conformation on the kinetics and the specificity of protein cleavage at neutral pH by the palladium aqua complexes. The scissile segments will then be inserted into fusion proteins tp demonstrate that these reagents can successfully cleave fusion proteins, in cases in which common proteolytic enzymes are inadequate; (2) develop two classes of new palladium-organic conjugate reagents, in which the ability of the Pd aqua complexes to cleave the polypeptide backbone is combined with the ability of one or two attached beta-cyclodextrins to recognize aromatic side chains proximal to the cleavage site. The stereochemical requirements for these simultaneous recognition-cleavage interactions will be investigated and used in designing optimal reagents for cleaving the x-Pro bond in the X-x-Pro-y-Y segments in which X or Y or both are phenylalanine, tyrosine, or tryptophan; (3) solve the problem of product inhibition and achieve catalytic turnover in the hydrolytic cleavage at neutral pH of the x-Pro bond in the X-x-Pro-y-Y segments in which both X and Y are aromatic residues with the Pd-bis(beta-cyclodextrin) reagent. Through a partnership with Invitrogen Co., which will endeavor to commercialize some of the products of this research, students will learn about industrial science, commercialization, and potential employment. This research will be widely disseminated in leading journals, seminars and international meetings, and in a new graduate special-topics course for chemists, biochemists and molecular biologists.