Kuhlman, B.

Biochemistry 2012, 51, 3933-3940, 10.1021/bi201881p

Here we show that a recent computationally designed zinc-mediated protein interface is serendipitously capable of catalyzing carboxyester and phosphoester hydrolysis. Although the original motivation was to design a de novo zinc-mediated protein–protein interaction (called MID1-zinc), we observed in the homodimer crystal structure a small cleft and open zinc coordination site. We investigated if the cleft and zinc site at the designed interface were sufficient for formation of a primitive active site that can perform hydrolysis. MID1-zinc hydrolyzes 4-nitrophenyl acetate with a rate acceleration of 105 and a kcat/KM of 630 M–1 s–1 and 4-nitrophenyl phosphate with a rate acceleration of 104 and a kcat/KM of 14 M–1 s–1. These rate accelerations by an unoptimized active site highlight the catalytic power of zinc and suggest that the clefts formed by protein–protein interactions are well-suited for creating enzyme active sites. This discovery has implications for protein evolution and engineering: from an evolutionary perspective, three-coordinated zinc at a homodimer interface cleft represents a simple evolutionary path to nascent enzymatic activity; from a protein engineering perspective, future efforts in de novo design of enzyme active sites may benefit from exploring clefts at protein interfaces for active site placement.

Pecoraro, V.L.

Biochemistry 2014, 53, 957-978, 10.1021/bi4016617

Zinc is an essential element required for the function of more than 300 enzymes spanning all classes. Despite years of dedicated study, questions regarding the connections between primary and secondary metal ligands and protein structure and function remain unanswered, despite numerous mechanistic, structural, biochemical, and synthetic model studies. Protein design is a powerful strategy for reproducing native metal sites that may be applied to answering some of these questions and subsequently generating novel zinc enzymes. From examination of the earliest design studies introducing simple Zn(II)-binding sites into de novo and natural protein scaffolds to current studies involving the preparation of efficient hydrolytic zinc sites, it is increasingly likely that protein design will achieve reaction rates previously thought possible only for native enzymes. This Current Topic will review the design and redesign of Zn(II)-binding sites in de novo-designed proteins and native protein scaffolds toward the preparation of catalytic hydrolytic sites. After discussing the preparation of Zn(II)-binding sites in various scaffolds, we will describe relevant examples for reengineering existing zinc sites to generate new or altered catalytic activities. Then, we will describe our work on the preparation of a de novo-designed hydrolytic zinc site in detail and present comparisons to related designed zinc sites. Collectively, these studies demonstrate the significant progress being made toward building zinc metalloenzymes from the bottom up.

Craik, C.S.

Biochemistry 1995, 34, 2172-2180, 10.1021/bi00007a010

Histidine substrate specificity has been engineered into trypsin by creating metal binding sites for Ni2+ and Zn2+ ions. The sites bridge the substrate and enzyme on the leaving-group side of the scissile bond. Application of simple steric and geometric criteria to a crystallographically derived enzyme- substrate model suggested that histidine specificity at the P2' position might be acheived by a tridentate site involving amino acid residues 143 and 151 of trypsin. Trypsin N143H/E151H hydrolyzes a P2'- His-containing peptide (AGPYAHSS) exclusively in the presence of nickel or zinc with a high level of catalytic efficiency. Since cleavage following the tyrosine residue is normally highly disfavored by trypsin, this result demonstrates that a metal cofactor can be used to modulate specificity in a designed fashion. The same geometric criteria applied in the primary SI binding pocket suggested that the single-site mutation D189H might effect metal-dependent His specificity in trypsin. However, kinetic and crystallographic analysis of this variant showed that the design was unsuccessful because His 189 rotates away from substrate causing a large perturbation in adjacent surface loops. This observation suggests that the reason specificity modification at the trypsin S1 site requires extensive mutagenesis is because the pocket cannot deform locally to accommodate alternate PI side chains. By taking advantage of the extended subsites, an alternate substrate specificity has been engineered into trypsin.

Lubitz, W.; Reijerse, E.

Biochemistry 2015, 54, 1474-1483, 10.1021/bi501391d

[FeFe]-hydrogenases are to date the only enzymes for which it has been demonstrated that the native inorganic binuclear cofactor of the active site Fe2(adt)(CO)3(CN)2 (adt = azadithiolate = [S-CH2-NH-CH2-S]2–) can be synthesized on the laboratory bench and subsequently inserted into the unmaturated enzyme to yield fully functional holo-enzyme (Berggren, G. et al. (2013) Nature 499, 66–70; Esselborn, J. et al. (2013) Nat. Chem. Biol. 9, 607–610). In the current study, we exploit this procedure to introduce non-native cofactors into the enzyme. Mimics of the binuclear subcluster with a modified bridging dithiolate ligand (thiodithiolate, N-methylazadithiolate, dimethyl-azadithiolate) and three variants containing only one CN– ligand were inserted into the active site of the enzyme. We investigated the activity of these variants for hydrogen oxidation as well as proton reduction and their structural accommodation within the active site was analyzed using Fourier transform infrared spectroscopy. Interestingly, the monocyanide variant with the azadithiolate bridge showed ∼50% of the native enzyme activity. This would suggest that the CN– ligands are not essential for catalytic activity, but rather serve to anchor the binuclear subsite inside the protein pocket through hydrogen bonding. The inserted artificial cofactors with a propanedithiolate and an N-methylazadithiolate bridge as well as their monocyanide variants also showed residual activity. However, these activities were less than 1% of the native enzyme. Our findings indicate that even small changes in the dithiolate bridge of the binuclear subsite lead to a rather strong decrease of the catalytic activity. We conclude that both the Brønsted base function and the conformational flexibility of the native azadithiolate amine moiety are essential for the high catalytic activity of the native enzyme.