Design of a Switchable Eliminase
Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 6823-6827, 10.1073/pnas.1018191108
The active sites of enzymes are lined with side chains whose dynamic, geometric, and chemical properties have been finely tuned relative to the corresponding residues in water. For example, the carboxylates of glutamate and aspartate are weakly basic in water but become strongly basic when dehydrated in enzymatic sites. The dehydration of the carboxylate, although intrinsically thermodynamically unfavorable, is achieved by harnessing the free energy of folding and substrate binding to reach the required basicity. Allosterically regulated enzymes additionally rely on the free energy of ligand binding to stabilize the protein in a catalytically competent state. We demonstrate the interplay of protein folding energetics and functional group tuning to convert calmodulin (CaM), a regulatory binding protein, into AlleyCat, an allosterically controlled eliminase. Upon binding Ca(II), native CaM opens a hydrophobic pocket on each of its domains. We computationally identified a mutant that (i) accommodates carboxylate as a general base within these pockets, (ii) interacts productively in the Michaelis complex with the substrate, and (iii) stabilizes the transition state for the reaction. Remarkably, a single mutation of an apolar residue at the bottom of an otherwise hydrophobic cavity confers catalytic activity on calmodulin. AlleyCat showed the expected pH-rate profile, and it was inactivated by mutation of its active site Glu to Gln. A variety of control mutants demonstrated the specificity of the design. The activity of this minimal 75-residue allosterically regulated catalyst is similar to that obtained using more elaborate computational approaches to redesign complex enzymes to catalyze the Kemp elimination reaction.
Metal: CaLigand type: Amino acidHost protein: C-terminal domain of calmodulinAnchoring strategy: DativeOptimization: GeneticReaction: Kemp eliminationMax TON: >40ee: ---PDB: 2KZ2Notes: Ca acts as allosteric regulator, catalytically active site contains no metal
Generation of a Hybrid Sequence-Specific Single Stranded Deoxyribonuclease
Science 1987, 238, 1401-1403, 10.1126/science.3685986
The relatively nonspecific single-stranded deoxyribonuclease, staphylococcal nuclease, was selectively fused to an oligonucleotide binding site of defined sequence to generate a hybrid enzyme. A cysteine was substituted for Lys116 in the enzyme by oligonucleotide-directed mutagenesis and coupled to an oligonucleotide that contained a 3'-thiol. The resulting hybrid enzyme cleaved single-stranded DNA at sites adjacent to the oligonucleotide binding site.
Metal: CaLigand type: UndefinedHost protein: Staphylococcal nucleaseAnchoring strategy: ---Optimization: ---Reaction: Hydrolytic cleavageMax TON: <1ee: ---PDB: ---Notes: Engineered sequence specificity
Metal Incorporated Horseradish Peroxidase (HRP) Catalyzed Oxidation of Resveratrol: Selective Dimerization or Decomposition
RSC Adv. 2013, 3, 22976, 10.1039/c3ra43784a
Horseradish Peroxidase (HRP) is a commercially available and prevalently used peroxidase with no specific substrate binding domain. However, after being incorporated with different metal cations, new catalytic functions were found in biomimetic oxidation of resveratrol. Based on the results of screening, Ca, Cu, Fe and Mn incorporated enzymes showed distinctive effects, either decomposition or dimerization products were observed.