Proton affinity of the oxyanion hole in the active site of ketosteroid isomerase.
The absorption spectra of a series of inhibitors bound at the active site of Delta(5)-3-ketosteroid isomerase from Pseudomonas putida were found to exhibit substantial variations in the contributions of the protonated and deprotonated forms. Systematic variation of the inhibitor solution pK(a) combined with a method of quantifying the contributions of each protonation state showed the oxyanion hole in the active site of wild-type Delta(5)-3-ketosteroid isomerase to have a proton affinity equal to a solution pK(a) of 10.05 +/- 0.03, which is similar to the measured pK(a) (10.0) of the reaction intermediate. This observation supports the prediction of Cleland, Kreevoy, Frey, Gassman, and Gerlt that an enzyme utilizing a strong hydrogen bond for catalysis matches the proton affinity of the protein to the intermediate [Cleland, W. W., and Kreevoy, M. M. (1994) Science 264, 1887-1890; Frey, P. A., Whitt, S., and Tobin, J. (1994) Science 264, 1927-1930; Gerlt, J. A., and Gassman, P. G. (1993) Biochemistry 32, 11934-11952]. As the difference in proton affinity decreases, the strength of the hydrogen bond increases, and the closely matched proton affinity between the active site and the reaction intermediate supports the possibility that a short, strong hydrogen bond is catalytically relevant in Delta(5)-3-ketosteroid isomerase.
Childs
William
W
Department of Chemistry, Stanford University, Stanford, California 94305-5080, USA.
Boxer
Steven G
SG
D002384:Catalysis; D020134:Catalytic Domain / Q000187:drug effects; D019295:Computational Biology; D018360:Crystallography, X-Ray; D006860:Hydrogen Bonding; D007664:Ketosteroids / Q000737:chemistry*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D011522:Protons; D016958:Pseudomonas putida / Q000201:enzymology; D055672:Static Electricity; D013251:Steroid Isomerases / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D007664:Ketosteroids; D011522:Protons; D013251:Steroid Isomerases; C022660:steroid delta-isomerase