Natural metalloenzymes are often the most proficient catalysts in terms of their activity, selectivity, and ability to operate at mild conditions. However, metalloenzymes are occasionally surprising in their selection of catalytic metals, and in their responses to metal substitution. Indeed, from the isolated standpoint of producing the best catalyst, a chemist designing from first-principles would likely choose a different metal. For example, some enzymes employ a redox active metal where a simple Lewis acid is needed. Such are several hydrolases. In other cases, substitution of a non-native metal leads to radical improvements in reactivity. For example, histone deacetylase 8 naturally operates with Zn(2+) in the active site but becomes much more active with Fe(2+). For β-lactamases, the replacement of the native Zn(2+) with Ni(2+) was suggested to lead to higher activity as predicted computationally. There are also intriguing cases, such as Fe(2+)- and Mn(2+)-dependent ribonucleotide reductases and W(4+)- and Mo(4+)-dependent DMSO reductases, where organisms manage to circumvent the scarcity of one metal (e.g., Fe(2+)) by creating protein structures that utilize another metal (e.g., Mn(2+)) for the catalysis of the same reaction. Naturally, even though both metal forms are active, one of the metals is preferred in every-day life, and the other metal variant remains dormant until an emergency strikes in the cell. These examples lead to certain questions. When are catalytic metals selected purely for electronic or structural reasons, implying that enzymatic catalysis is optimized to its maximum? When are metal selections a manifestation of competing evolutionary pressures, where choices are dictated not just by catalytic efficiency but also by other factors in the cell? In other words, how can enzymes be improved as catalysts merely through the use of common biological building blocks available to cells? Addressing these questions is highly relevant to the enzyme design community, where the goal is to prepare maximally efficient quasi-natural enzymes for the catalysis of reactions that interest humankind. Due to competing evolutionary pressures, many natural enzymes may not have evolved to be ideal catalysts and can be improved for the isolated purpose of catalysis in vitro when the competing factors are removed. The goal of this Account is not to cover all the possible stories but rather to highlight how variable enzymatic catalysis can be. We want to bring up possible factors affecting the evolution of enzyme structure, and the large- and intermediate-scale structural and electronic effects that metals can induce in the protein, and most importantly, the opportunities for optimization of these enzymes for catalysis in vitro.

Histone deacetylases (HDACs) are responsible for the removal of acetyl groups from histones, resulting in gene silencing. Overexpression of HDACs is associated with cancer, and their inhibitors are of particular interest as chemotherapeutics. However, HDACs remain a target of mechanistic debate. HDAC class 8 is the most studied HDAC, and of particular importance due to its human oncological relevance. HDAC8 has traditionally been considered to be a Zn-dependent enzyme. However, recent experimental assays have challenged this assumption and shown that HDAC8 is catalytically active with a variety of different metals, and that it may be a Fe-dependent enzyme in vivo. We studied two opposing mechanisms utilizing a series of divalent metal ions in physiological abundance (Zn(2+), Fe(2+), Co(2+), Mn(2+), Ni(2+), and Mg(2+)). Extensive sampling of the entire protein with different bound metals was done with the mixed quantum-classical QM/DMD method. Density functional theory (DFT) on an unusually large cluster model was used to describe the active site and reaction mechanism. We have found that the reaction profile of HDAC8 is similar among all metals tested, and follows one of the previously published mechanisms, but the rate-determining step is different from the one previously claimed. We further provide a scheme for estimating the metal binding affinities to the protein. We use the quantum theory of atoms in molecules (QTAIM) to understand the different binding affinities for each metal in HDAC8 as well as the ability of each metal to bind and properly orient the substrate for deacetylation. The combination of this data with the catalytic rate constants is required to reproduce the experimentally observed trend in metal-depending performance. We predict Co(2+) and Zn(2+) to be the most active metals in HDAC8, followed by Fe(2+), and Mn(2+) and Mg(2+) to be the least active.

Combined inhibition of ribonucleotide reductase and deoxycytidine kinase (dCK) in multiple cancer cell lines depletes deoxycytidine triphosphate pools leading to DNA replication stress, cell cycle arrest, and apoptosis. Evidence implicating dCK in cancer cell proliferation and survival stimulated our interest in developing small molecule dCK inhibitors. Following a high throughput screen of a diverse chemical library, a structure-activity relationship study was carried out. Positron Emission Tomography (PET) using (18)F-L-1-(2'-deoxy-2'-FluoroArabinofuranosyl) Cytosine ((18)F-L-FAC), a dCK-specific substrate, was used to rapidly rank lead compounds based on their ability to inhibit dCK activity in vivo. Evaluation of a subset of the most potent compounds in cell culture (IC50 = ∼1-12 nM) using the (18)F-L-FAC PET pharmacodynamic assay identified compounds demonstrating superior in vivo efficacy.

Combined inhibition of ribonucleotide reductase and deoxycytidine kinase (dCK) in multiple cancer cell lines depletes deoxycytidine triphosphate pools leading to DNA replication stress, cell cycle arrest, and apoptosis. Evidence implicating dCK in cancer cell proliferation and survival stimulated our interest in developing small molecule dCK inhibitors. Following a high throughput screen of a diverse chemical library, a structure-activity relationship study was carried out. Positron Emission Tomography (PET) using (18)F-L-1-(2'-deoxy-2'-FluoroArabinofuranosyl) Cytosine ((18)F-L-FAC), a dCK-specific substrate, was used to rapidly rank lead compounds based on their ability to inhibit dCK activity in vivo. Evaluation of a subset of the most potent compounds in cell culture (IC50 = ∼1-12 nM) using the (18)F-L-FAC PET pharmacodynamic assay identified compounds demonstrating superior in vivo efficacy.