PMC

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

Kinetic model used to analyze the activity of PZD2.

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

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.

(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.

Molecular surfaces () focusing on the active site of PZD2 with substrate atoms in green (A) and the corresponding region in the x-ray crystal structure () 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.

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 () in all spectra and in combination with free and acylated protein results in multiple peaks.