Proton affinity of the oxyanion hole in the active site of ketosteroid isomerase

Abstract

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.

Figure 1

Schematic of enzymatic isomerization of a Δ⁵-3-ketosteroid to a Δ⁴-3-ketosteroid as observed in KSI from Pseudomonas putida and Comamonas testosteroni. Residues are numbered according to the P. putida sequence. In C. testosteroni, Tyr16 is Tyr14, Asp103 is Asp99, and Asp40 is Asp38.

Figure 2

Figure 3

The absorption spectra of substituted naphthols (Black, normalized at the absorption maximum) fit to a linear combination (Red) of the protonated (Blue) and deprotonated (Green) species, each shifted as described in text and in Table 1. The protonated and deprotonated spectra are scaled by their relative contributions to the fit (Table 1). The scaling factors are shown in the color corresponding to the curve they describe. (A) 2,6-dihydroxynaphthalene, solution pKa 10.1; (B) 6-methoxy-2-naphthol, solution pKa 9.89; (C) equilenin, solution pKa 9.7; (D) 6-bromo-2-naphthol, solution pKa 9.42; and (E) 6-cyano-2-naphthol, solution pKa 8.4.

Figure 4

Fraction of substrate bound as the protonated species (Table 1) versus the substrate pK_a (black dots) is fit to Equation 4 (red line) to determine the proton affinity of the active site. Error bars indicate one standard deviation.

Figure 5

The absorption spectrum of equilenin bound to WT-pKSI (Black) fit to a linear combination (Red) of the protonated (Blue) and deprotonated (Green) species. The protonated and deprotonated spectra are scaled by their relative contributions to the fit, and the scaling factors are shown below each curve.