De novo Designed Metalloenzymes:Structural Stabilization and Hydrolytic Catalysis in a Family of-helical Coiled Coils.

Metalloprotein Design;Chemistry;Science;Chemistry

Zastrow, Melissa LynnAl-Hashimi, Hashim M. ;

University of Michigan

关键词: Metalloprotein Design;    Chemistry;    Science;    Chemistry;   

Others  :  https://deepblue.lib.umich.edu/bitstream/handle/2027.42/102295/zastrowm_1.pdf?sequence=3&isAllowed=y

瑞士|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Fundamental studies on structural stabilization and hydrolytic catalysis in a family of de novo designed alpha-helical three-stranded coiled coils are presented. Basic principles are used to discriminate structural and catalytic sites (Hg(II)Cys3 and Zn(II)His3). Spectroscopy, X-ray crystallography, and kinetic studies are exploited to characterize these sites.X-ray crystal structures of these metallopeptides ([Hg(II)]S(Zn(II)(OH2/OH-)]N(CSL9PenL23H)3n+) are determined to 2.2 Å resolution (pH 7.5 and 8.5). Both structures contain a tetrahedral ZnN3X site, like the active site of carbonic anhydrase (CA). At low pH a mixture of HgS2 and HgS3 centers is observed, while the high pH form exclusively shows HgS3. Denaturation studies verify the HgS3 site provides structural stability, while kinetic analysis of [Hg(II)]S[Zn(II)(OH2/OH-)]N(TRIL9CL23H)3n+ towards non-physiological substrate, p-nitrophenyl acetate (pNPA), confirms the ZnN3O site is hydrolytically active with a maximal efficiency (31 M-1 s-1) only ~100-fold less than the fastest CA and pKa ~2 units higher (9.0 vs 6.8). More significantly, analysis towards physiological substrate (CO2) confirms the design of a minimal Zn(II)-binding site with a catalytic efficiency (~2.6 x 10^5 M-1 s-1) within ~400-fold of a highly evolved enzyme. This minimal active site structure becomes even more competitive with CAII mutants that exclude important secondary hydrogen-bonding interactions.Variation of the position of the ZnN3O site along the sequence of the peptides (TRIL9HL23C, TRIL9CL19H) and insertion into a longer, more stable sequence (GrandL2WL16CL30H) demonstrates that while location can be used to control binding affinity, solvent, substrate, and inhibitor access, Zn(II)-OH2 pKa, and catalytic rate, the maximal catalytic efficiency remains significantly high. Kinetic analysis in the absence of the structural site ([Zn(II)(OH2/OH-)]N(TRIL2WL23H)3n+) yields similar parameters demonstrating that this extra stability is not detrimental to the function of the second site.Polar residues (Thr, Asp) are incorporated into Grand (GrandL16CL26TL30H, GrandL16CL26DL30H) for implementing secondary interactions involving hydrogen bonding in order to fine-tune the designed hydrolytic site. These substitutions do not result in large changes to the catalytic efficiency; however, Zn(II)-binding affinities increase ~70-fold. Co(II) binding (as a spectroscopic probe) to these variants indicates different and probably mixed coordination geometries relative to [Co(II)(OH2/OH-)x]N (TRIL2WL23H)3n+, supporting the need for asymmetric site design in future work.