5KP3
Crystal Structure of Ketosteroid Isomerase from Pseudomonas putida (pKSI) bound to Equilenin; D40N, Y57(Cl-Y)
Summary for 5KP3
| Entry DOI | 10.2210/pdb5kp3/pdb |
| Related | 5D81 5D82 5KP1 5KP4 |
| Descriptor | Steroid Delta-isomerase, EQUILENIN, SULFATE ION, ... (4 entities in total) |
| Functional Keywords | isomerase |
| Biological source | Pseudomonas putida |
| Total number of polymer chains | 2 |
| Total formula weight | 30821.91 |
| Authors | Wu, Y.,Boxer, S.G. (deposition date: 2016-07-01, release date: 2016-09-07, Last modification date: 2025-04-02) |
| Primary citation | Wu, Y.,Boxer, S.G. A Critical Test of the Electrostatic Contribution to Catalysis with Noncanonical Amino Acids in Ketosteroid Isomerase. J.Am.Chem.Soc., 138:11890-11895, 2016 Cited by PubMed Abstract: The vibrational Stark effect (VSE) has been used to measure the electric field in the active site of ketosteroid isomerase (KSI). These measured fields correlate with ΔG(⧧) in a series of conventional mutants, yielding an estimate for the electrostatic contribution to catalysis (Fried et al. Science 2014, 346, 1510-1513). In this work we test this result with much more conservative variants in which individual Tyr residues in the active site are replaced by 3-chlorotyrosine via amber suppression. The electric fields sensed at the position of the carbonyl bond involved in charge displacement during catalysis were characterized using the VSE, where the field sensitivity has been calibrated by vibrational Stark spectroscopy, solvatochromism, and MD simulations. A linear relationship is observed between the electric field and ΔG(⧧) that interpolates between wild-type and more drastic conventional mutations, reinforcing the evaluation of the electrostatic contribution to catalysis in KSI. A simplified model and calculation are developed to estimate changes in the electric field accompanying changes in the extended hydrogen-bond network in the active site. The results are consistent with a model in which the O-H group of a key active site tyrosine functions by imposing a static electrostatic potential onto the carbonyl bond. The model suggests that the contribution to catalysis from the active site hydrogen bonds is of similar weight to the distal interactions from the rest of the protein. A similar linear correlation was also observed between the proton affinity of KSI's active site and the catalytic rate, suggesting a direct connection between the strength of the H-bond and the electric field it exerts. PubMed: 27545569DOI: 10.1021/jacs.6b06843 PDB entries with the same primary citation |
| Experimental method | X-RAY DIFFRACTION (1.7 Å) |
Structure validation
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