Enzyme-like proteins by computational design

Abstract

We report the development and initial experimental validation of a computational design procedure aimed at generating enzyme-like protein catalysts called "protozymes." Our design approach utilizes a "compute and build" strategy that is based on the physical/chemical principles governing protein stability and catalytic mechanism. By using the catalytically inert 108-residue Escherichia coli thioredoxin as a scaffold, the histidine-mediated nucleophilic hydrolysis of p-nitrophenyl acetate as a model reaction, and the ORBIT protein design software to compute sequences, an active site scan identified two promising catalytic positions and surrounding active-site mutations required for substrate binding. Experimentally, both candidate protozymes demonstrated catalytic activity significantly above background. One of the proteins, PZD2, displayed "burst" phase kinetics at high substrate concentrations, consistent with the formation of a stable enzyme intermediate. The kinetic parameters of PZD2 are comparable to early catalytic Abs. But, unlike catalytic Ab design, our design procedure is independent of fold, suggesting a possible mechanism for examining the relationships between protein fold and the evolvability of protein function.

Figure 1

(A) Nucleophile-mediated catalysis of PNPA hydrolysis. (B) High-energy state structure used in the computational active site scan. Labeled dihedral angles were varied to generate the set of high-energy state rotamers used in the design calculations.

Figure 2

Molecular surfaces (43) focusing on the active site of PZD2 with substrate atoms in green (A) and the corresponding region in the x-ray crystal structure (38) of the wild-type scaffold (B and C). An active-site cleft is present in the design of PZD2 that is largely filled in the wild-type structure. Wild-type residues that were mutated to create the active site are shown in C (F12, L17, and Y70). In the design of PZD2, all side chains were allowed to change geometry, resulting in a slightly different surface compared with that of the wild-type protein.

Figure 3

Kinetic model used to analyze the activity of PZD2.

Figure 4

Velocity vs. substrate concentration for the hydrolysis of PNPA by PZD2.

Figure 5

Buffer-corrected hydrolysis of PNPA by PZD2 (●), PZD2 H17A (□), wild-type thioredoxin (▴), and wild-type L17H/D26I (×). Data are shown for high substrate concentration and equivalent low protein concentration.

Figure 6

Trapping of an acylated intermediate by mass spectrometry. (A) PZD2, (B) PZD2 reacted with substrate, (C) PZD2 H17A, and (D) PZD2 H17A reacted with substrate. A large increase in the population of a +42 species occurs on reaction of PZD2 with substrate, indicating the buildup of an acyl-enzyme intermediate. This +42 species is dramatically reduced for PZD2 H17A where the designed catalytic histidine was mutated to alanine. A small increase in the population of a +42 species is detected in PZD2 H17A on reaction with substrate and is likely the result of acylation at the single surface-exposed histidine at position 6. Consistent with this analysis, a small increase in the population of a double acetylated +84 product is detected on reaction of PZD2 with substrate. A copper matrix adduct (+63) is present (38) in all spectra and in combination with free and acylated protein results in multiple peaks.

Figure 7

Lineweaver–Burk analysis of PZD2-catalyzed PNPA hydrolysis in the presence (□) and absence (●) of 10 mM PNPG.