Hydrogen bonding in the active site of ketosteroid isomerase: electronic inductive effects and hydrogen bond coupling.
Computational studies are performed to analyze the physical properties of hydrogen bonds donated by Tyr16 and Asp103 to a series of substituted phenolate inhibitors bound in the active site of ketosteroid isomerase (KSI). As the solution pK(a) of the phenolate increases, these hydrogen bond distances decrease, the associated nuclear magnetic resonance (NMR) chemical shifts increase, and the fraction of protonated inhibitor increases, in agreement with prior experiments. The quantum mechanical/molecular mechanical calculations provide insight into the electronic inductive effects along the hydrogen bonding network that includes Tyr16, Tyr57, and Tyr32, as well as insight into hydrogen bond coupling in the active site. The calculations predict that the most-downfield NMR chemical shift observed experimentally corresponds to the Tyr16-phenolate hydrogen bond and that Tyr16 is the proton donor when a bound naphtholate inhibitor is observed to be protonated in electronic absorption experiments. According to these calculations, the electronic inductive effects along the hydrogen bonding network of tyrosines cause the Tyr16 hydroxyl to be more acidic than the Asp103 carboxylic acid moiety, which is immersed in a relatively nonpolar environment. When one of the distal tyrosine residues in the network is mutated to phenylalanine, thereby diminishing this inductive effect, the Tyr16-phenolate hydrogen bond becomes longer and the Asp103-phenolate hydrogen bond shorter, as observed in NMR experiments. Furthermore, the calculations suggest that the differences in the experimental NMR data and electronic absorption spectra for pKSI and tKSI, two homologous bacterial forms of the enzyme, are due predominantly to the third tyrosine that is present in the hydrogen bonding network of pKSI but not tKSI. These studies also provide experimentally testable predictions about the impact of mutating the distal tyrosine residues in this hydrogen bonding network on the NMR chemical shifts and electronic absorption spectra.
Department of Chemistry,Pennsylvania State University, University Park, PA 16802, USA.
Philip
P
Hanoian
Paul A
PA
Sigala
Daniel
D
Herschlag
Sharon
S
Hammes-Schiffer
D019943:Amino Acid Substitution; D020134:Catalytic Domain; D020604:Comamonas testosteroni / Q000201:enzymology; D004583:Electrons; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D006860:Hydrogen Bonding; D062385:Hydroxybenzoates / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D007536:Isomerism; D007664:Ketosteroids / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011522:Protons; D016958:Pseudomonas putida / Q000201:enzymology; D011789:Quantum Theory; D013251:Steroid Isomerases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D004791:Enzyme Inhibitors; D062385:Hydroxybenzoates; D007664:Ketosteroids; D011522:Protons; D013251:Steroid Isomerases; C017616:phenolic acid