17 publications

17 publications

A Cofactor Approach to Copper-Dependent Catalytic Antibodies

Janda, K. D.; Nicholas, K. M.

Proc. Natl. Acad. Sci. U. S. A., 2002, 10.1073/pnas.052001099

A strategy for the preparation of semisynthetic copper(II)-based catalytic metalloproteins is described in which a metal-binding bis-imidazole cofactor is incorporated into the combining site of the aldolase antibody 38C2. Antibody 38C2 features a large hydrophobic-combining site pocket with a highly nucleophilic lysine residue, LysH93, that can be covalently modified. A comparison of several lactone and anhydride reagents shows that the latter are the most effective and general derivatizing agents for the 38C2 Lys residue. A bis-imidazole anhydride (5) was efficiently prepared from N-methyl imidazole. The 38C2–5-Cu conjugate was prepared by either (i) initial derivatization of 38C2 with 5 followed by metallation with CuCl2, or (ii) precoordination of 5 with CuCl2 followed by conjugation with 38C2. The resulting 38C2–5-Cu conjugate was an active catalyst for the hydrolysis of the coordinating picolinate ester 11, following Michaelis–Menten kinetics [kcat(11) = 2.3 min−1 and Km(11) 2.2 mM] with a rate enhancement [kcat(11)kuncat(11)] of 2.1 × 105. Comparison of the second-order rate constants of the modified 38C2 and the Cu(II)-bis-imidazolyl complex k(6-CuCl2) gives a rate enhancement of 3.5 × 104 in favor of the antibody complex with an effective molarity of 76.7 M, revealing a significant catalytic benefit to the binding of the bis-imidazolyl ligand into 38C2.


Metal: Cu
Ligand type: Bisimidazol
Host protein: Antibody 38C2
Anchoring strategy: Covalent
Optimization: Genetic
Max TON: ---
ee: ---
PDB: ---
Notes: ---

A Designed Supramolecular Protein Assembly with In Vivo Enzymatic Activity

Tezcan, F. A.

Science, 2014, 10.1126/science.1259680

The generation of new enzymatic activities has mainly relied on repurposing the interiors of preexisting protein folds because of the challenge in designing functional, three-dimensional protein structures from first principles. Here we report an artificial metallo-β-lactamase, constructed via the self-assembly of a structurally and functionally unrelated, monomeric redox protein into a tetrameric assembly that possesses catalytic zinc sites in its interfaces. The designed metallo-β-lactamase is functional in the Escherichia coli periplasm and enables the bacteria to survive treatment with ampicillin. In vivo screening of libraries has yielded a variant that displays a catalytic proficiency [(kcat/Km)/kuncat] for ampicillin hydrolysis of 2.3 × 106 and features the emergence of a highly mobile loop near the active site, a key component of natural β-lactamases to enable substrate interactions.


Metal: Zn
Ligand type: Amino acid
Host protein: Cytochrome cb562
Anchoring strategy: Dative
Optimization: Genetic
Max TON: ---
ee: ---
PDB: 4U9E
Notes: ---

A Highly Specific Metal-Activated Catalytic Antibody

Janda, K. D.; Lerner, R. A.

J. Am. Chem. Soc., 1993, 10.1021/ja00064a068

n/a


Metal: Zn
Ligand type: Undefined
Host protein: IgG 84A3
Anchoring strategy: Undefined
Optimization: ---
Max TON: ---
ee: ---
PDB: ---
Notes: Substrate specificty

Artificial Metalloenzymes based on Protein Cavities: Exploring the Effect of Altering the Metal Ligand Attachment Position by Site Directed Mutagenesis

Distefano, M. D.

Bioorg. Med. Chem. Lett., 1999, 10.1016/S0960-894X(98)00684-2


Metal: Cu
Ligand type: Phenanthroline
Anchoring strategy: Covalent
Optimization: Genetic
Max TON: 1 to 4
ee: 61 to 94
PDB: ---
Notes: Varied attachment position

A Semisynthetic Metalloenzyme based on a Protein Cavity that Catalyzes the Enantioselective Hydrolysis of Ester and Amide Substrates

Distefano, M. D.

J. Am. Chem. Soc., 1997, 10.1021/JA970820K

In an effort to prepare selective and efficient catalysts for ester and amide hydrolysis, we are designing systems that position a coordinated metal ion within a defined protein cavity. Here, the preparation of a protein-1,10-phenanthroline conjugate and the hydrolytic chemistry catalyzed by this construct are described. Iodoacetamido-1,10-phenanthroline was used to modify a unique cysteine residue in ALBP (adipocyte lipid binding protein) to produce the conjugate ALBP-Phen. The resulting material was characterized by electrospray mass spectrometry, UV/vis and fluorescence spectroscopy, gel filtration chromatography, and thiol titration. The stability of ALBP-Phen was evaluated by guanidine hydrochloride denaturation experiments, and the ability of the conjugate to bind Cu(II) was demonstrated by fluorescence spectroscopy. ALBP-Phen-Cu(II) catalyzes the enantioselective hydrolysis of several unactivated amino acid esters under mild conditions (pH 6.1, 25 °C) at rates 32−280-fold above the background rate in buffered aqueous solution. In 24 h incubations 0.70 to 7.6 turnovers were observed with enantiomeric excesses ranging from 31% ee to 86% ee. ALBP-Phen-Cu(II) also promotes the hydrolysis of an aryl amide substrate under more vigorous conditions (pH 6.1, 37 °C) at a rate 1.6 × 104-fold above the background rate. The kinetics of this amide hydrolysis reaction fit the Michaelis−Menten relationship characteristic of enzymatic processes. The rate enhancements for ester and amide hydrolysis reported here are 102−103 lower than those observed for free Cu(II) but comparable to those previously reported for Cu(II) complexes.


Metal: Cu
Ligand type: Phenanthroline
Anchoring strategy: Covalent
Optimization: ---
Max TON: 1 to 8
ee: 39 to 86
PDB: ---
Notes: ---

Catalysis by a De Novo Zinc-Mediated Protein Interface: Implications for Natural Enzyme Evolution and Rational Enzyme Engineering

Kuhlman, B.

Biochemistry, 2012, 10.1021/bi201881p


Metal: Zn
Ligand type: Amino acid
Anchoring strategy: Dative
Optimization: Chemical & genetic
Max TON: >50
ee: ---
PDB: 3V1C
Notes: ---

Catalytic Properties and Specificity of the Extracellular Nuclease of Staphylococcus Aureus

Cuatrecasas, P.

J. Biol. Chem., 1967, PMID 4290246


Metal: Sr
Ligand type: Amino acid
Host protein: Nuclease from S. aureus
Anchoring strategy: Metal substitution
Optimization: ---
Max TON: ---
ee: ---
PDB: ---
Notes: DNA cleavage

Computational Redesign of a Mononuclear Zinc Metalloenzyme for Organophosphate Hydrolysis

Baker, D.

Nat. Chem. Biol., 2012, 10.1038/NChemBio.777


Metal: Zn
Ligand type: Amino acid
Anchoring strategy: Dative
Optimization: Genetic
Max TON: >140
ee: ---
PDB: 3T1G
Notes: kcat/KM ≈ 104 M-1*s-1

Design and Evolution of New Catalytic Activity with an Existing Protein Scaffold

Kim, H. S.

Science, 2006, 10.1126/science.1118953


Metal: Zn
Ligand type: Amino acid
Host protein: Glyoxalase II (Human)
Anchoring strategy: Dative
Optimization: Genetic
Max TON: ---
ee: ---
PDB: 2F50
Notes: kcat/KM ≈ 184 M-1*s-1

Engineered Metal Regulation of Trypsin Specificity

Craik, C. S.

Biochemistry, 1995, 10.1021/bi00007a010


Metal: Zn
Ligand type: Amino acid
Host protein: Trypsin
Anchoring strategy: Dative
Optimization: Genetic
Max TON: ---
ee: ---
PDB: ---
Notes: Substrate specificty

Metal: Ni
Ligand type: Amino acid
Host protein: Trypsin
Anchoring strategy: Dative
Optimization: Genetic
Max TON: ---
ee: ---
PDB: ---
Notes: Substrate specificty

Generation of a Hybrid Sequence-Specific Single Stranded Deoxyribonuclease

Schultz, P. G.

Science, 1987, 10.1126/science.3685986


Metal: Ca
Ligand type: Undefined
Host protein: Staphylococcal nuclease
Anchoring strategy: ---
Optimization: ---
Max TON: <1
ee: ---
PDB: ---
Notes: Engineered sequence specificity

Hydrolytic Catalysis and Structural Stabilization in a Designed Metalloprotein

Pecoraro, V. L.

Nat. Chem., 2011, 10.1038/NCHEM.1201


Metal: Hg; Zn
Ligand type: Amino acid
Host protein: TRI peptide
Anchoring strategy: Dative
Optimization: Chemical & genetic
Max TON: >10
ee: ---
PDB: 3PBJ
Notes: Zn ion for catalytic activity, Hg ion for structural stability of the ArM. PDB ID 3PBJ = Structure of an analogue.

Metal: Hg; Zn
Ligand type: Amino acid
Host protein: TRI peptide
Anchoring strategy: Dative
Optimization: Chemical & genetic
Max TON: ---
ee: ---
PDB: 3PBJ
Notes: Zn ion for catalytic activity, Hg ion for structural stability of the ArM, kcat/KM ≈ 1.8*105 M-1*s-1. PDB ID 3PBJ = Structure of an analogue.

Influence of Active Site Location on Catalytic Activity in De Novo-Designed Zinc Metalloenzymes

Pecoraro, V. L.

J. Am. Chem. Soc., 2013, 10.1021/ja401537t


Metal: Hg; Zn
Ligand type: Amino acid
Host protein: TRI peptide
Anchoring strategy: Dative
Optimization: Chemical & genetic
Max TON: ---
ee: ---
PDB: 3PBJ
Notes: Influence of position of Zn and Hg ion on catalytic activity of the ArM tested. PDB ID 3PBJ = Structure of an analogue.

Metal Ion Dependent Binding of Sulphonamide to Carbonic Anhydrase

Coleman, J. E.

Nature, 1967, 10.1038/214193a0


Metal: Co
Ligand type: Amino acid
Host protein: Human carbonic anhydrase
Anchoring strategy: Metal substitution
Optimization: ---
Max TON: ---
ee: ---
PDB: ---
Notes: CO2 hydration

Metal: Co
Ligand type: Amino acid
Host protein: Human carbonic anhydrase
Anchoring strategy: Metal substitution
Optimization: ---
Max TON: ---
ee: ---
PDB: ---
Notes: Ester cleavage

Neocarzinostatin-Based Hybrid Biocatalysts with a RNase like Activity

Mahy, J.-P.; Ricoux, R.

Bioorg. Med. Chem., 2014, 10.1016/j.bmc.2014.05.063


Metal: Zn
Ligand type: Poly-pyridine
Anchoring strategy: Supramolecular
Optimization: ---
Max TON: ---
ee: ---
PDB: ---
Notes: kcat/KM = 13.6 M-1 * s-1

Rare Earth Metal Ions as Probes of Calcium Binding Sites in Proteins: Neodynium Acceleration of the Activation of Trypsinogen

Birnbaum, E. R.; Darnall, D. W.

J. Biol. Chem., 1970, PMID 5484822


Metal: Nd
Ligand type: Amino acid
Host protein: Trypsin
Anchoring strategy: Metal substitution
Optimization: ---
Max TON: <1
ee: ---
PDB: ---
Notes: ---

Sequence-Specific Peptide Cleavage Catalyzed by an Antibody

Lerner, R. A.

Science, 1989, 10.1126/science.2922606


Metal: Zn
Ligand type: Tetramine
Host protein: Antibody 28F11
Anchoring strategy: Supramolecular
Optimization: Chemical
Max TON: 400
ee: ---
PDB: ---
Notes: ---