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. 2017 Jan 31;56(4):582-591.
doi: 10.1021/acs.biochem.6b00762. Epub 2017 Jan 20.

Kemp Eliminase Activity of Ketosteroid Isomerase

Vandana Lamba  1 , Enis Sanchez  2 , Lauren Rose Fanning  2 , Kathryn Howe  3 , Maria Alejandra Alvarez  4 , Daniel Herschlag  1   5 , Marcello Forconi  2
Affiliations

Kemp Eliminase Activity of Ketosteroid Isomerase

Vandana Lamba et al. Biochemistry. .

Abstract

Kemp eliminases represent the most successful class of computationally designed enzymes, with rate accelerations of up to 109-fold relative to the rate of the same reaction in aqueous solution. Nevertheless, several other systems such as micelles, catalytic antibodies, and cavitands are known to accelerate the Kemp elimination by several orders of magnitude. We found that the naturally occurring enzyme ketosteroid isomerase (KSI) also catalyzes the Kemp elimination. Surprisingly, mutations of D38, the residue that acts as a general base for its natural substrate, produced variants that catalyze the Kemp elimination up to 7000-fold better than wild-type KSI does, and some of these variants accelerate the Kemp elimination more than the computationally designed Kemp eliminases. Analysis of the D38N general base KSI variant suggests that a different active site carboxylate residue, D99, performs the proton abstraction. Docking simulations and analysis of inhibition by active site binders suggest that the Kemp elimination takes place in the active site of KSI and that KSI uses the same catalytic strategies of the computationally designed enzymes. In agreement with prior observations, our results strengthen the conclusion that significant rate accelerations of the Kemp elimination can be achieved with very few, nonspecific interactions with the substrate if a suitable catalytic base is present in a hydrophobic environment. Computational design can fulfill these requirements, and the design of more complex and precise environments represents the next level of challenges for protein design.

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Figures

Figure 1
Figure 1
(A) The Kemp elimination. X represents a generic substituent on the benzisoxazole ring, and B represent a base. (B) The reaction pathway for the KSI-catalyzed reaction, which involves a proton transfer reaction in the first step.
Figure 2
Figure 2
Initial velocities for the Kemp elimination in the presence (filled dots) and absence (empty dots) of wild type tKSI as a function of the concentration of 5-nitrobenzisoxazole (25 mM sodium HEPES, pH 6.97, 22 °C, [tKSI] = 5.7 μM). Points in the inset represent the difference between the rate of the reaction in the presence and in the absence of tKSI.
Figure 3
Figure 3
pH-rate profile for the reaction of wild type tKSI in the presence of 1.0 mM 5NBI, 5.7 μM tKSI, 25 mM buffer, 22 °C. Points represented by empty circles were fit to Equation 1, which describes a single deprotonation event. Empty squares represent measurements for which the hydroxide-catalyzed reaction is faster than the KSI-catalyzed reaction, and were excluded from the fit.
Figure 4
Figure 4
pH-rate profiles for the D38N tKSI-catalyzed Kemp eliminations of 5NBI (filled circles) and 5BrBI (filled squares) in 25 mM buffer, 22 °C. Fitting the data to Equation 1 gives pKa values of 7.8 ± 0.2 for 5NBI and 7.9 ± 0.2 for 5BrBI.
Figure 5
Figure 5
pH-rate profiles for the Kemp elimination of 4,6-Cl2BI catalyzed by D38N (empty circles) or D38N/D99N (empty squares). Lines represent a fit to Equation 1, which give pKa values of 8.0 ± 0.2 for D38N and > 9.0 for D38N/D99N.
Figure 6
Figure 6
Docking simulation of 5NBI binding to D38N tKSI (PDB ID 1OHP). Oxygen atoms are in red, nitrogen in blue, hydrogen in white, and carbon on the protein in magenta and carbon on 5NBI in green. (A) Lowest energy structure with residues within 4 Å of 5NBI shown as spheres. (B) Close-up view of the active site, with distances from the labile hydrogen of 5NBI to the nitrogen atom of D38 and to one of the oxygen atoms of D99.
Figure 6
Figure 6
Docking simulation of 5NBI binding to D38N tKSI (PDB ID 1OHP). Oxygen atoms are in red, nitrogen in blue, hydrogen in white, and carbon on the protein in magenta and carbon on 5NBI in green. (A) Lowest energy structure with residues within 4 Å of 5NBI shown as spheres. (B) Close-up view of the active site, with distances from the labile hydrogen of 5NBI to the nitrogen atom of D38 and to one of the oxygen atoms of D99.
Figure 7
Figure 7
Inhibition of the D38N tKSI-catalyzed Kemp elimination of 4,6-Cl2BI by 4-chlorophenol (4Cl-Ph). Reactions were carried out in the presence of 0.39 μM enzyme, 600 μM substrate, pH 7.5, and different concentrations of 4-ClPh. Points were fit to the equation v=vin+vmax×KIKI+[4-ClPh] where vin represents the velocity at full inhibition, vmax the velocity in absence of the inhibitor, and KI the inhibition constant.
Figure 8
Figure 8
Relationship between the natural logarithm of the second-order rate constant for the D38N tKSI-catalyzed Kemp elimination and the pKa of the product of the reaction. Reactions were carried out at pH 9.5 using subsaturating concentrations of benzisoxazoles.
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