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Crystal Structure of Δ5-3-Ketosteroid Isomerase from Pseudomonas testosteroni in Complex with Equilenin Settles the Correct Hydrogen Bonding Scheme for Transition State Stabilization*

Open AccessPublished:November 12, 1999DOI:https://doi.org/10.1074/jbc.274.46.32863
      Δ5-3-Ketosteroid isomerase from Pseudomonas testosteroni has been intensively studied as a prototype to understand an enzyme-catalyzed allylic isomerization. Asp38 (pK a ∼4.7) was identified as the general base abstracting the steroid C4β proton (pK a ∼12.7) to form a dienolate intermediate. A key and common enigmatic issue involved in the proton abstraction is the question of how the energy required for the unfavorable proton transfer can be provided at the active site of the enzyme and/or how the thermodynamic barrier can be drastically reduced. Answering this question has been hindered by the existence of two differently proposed enzyme reaction mechanisms. The 2.26 Å crystal structure of the enzyme in complex with a reaction intermediate analogue equilenin reveals clearly that both the Tyr14 OH and Asp99 COOH provide direct hydrogen bonds to the oxyanion of equilenin. The result negates the catalytic dyad mechanism in which Asp99 donates the hydrogen bond to Tyr14, which in turn is hydrogen bonded to the steroid. A theoretical calculation also favors the doubly hydrogen-bonded system over the dyad system. Proton nuclear magnetic resonance analyses of several mutant enzymes indicate that the Tyr14 OH forms a low barrier hydrogen bond with the dienolic oxyanion of the intermediate.
      KSI
      ketosteroid isomerase
      H-bond
      hydrogen bond
      LBHB
      low barrier hydrogen bond
      19-NTHS
      19-nortestosterone hemisuccinate
      rmsd
      root mean square difference
      Heterolytic C-H bond cleavage is a fundamental process found in a wide variety of biological reactions such as aldol/Claisen condensation, racemization, transamination, and isomerization reactions (
      • Walsh C.
      ,
      • Gerlt J.A.
      • Kozarich J.W.
      • Kenyon G.L.
      • Gassman P.G.
      ). In almost all the reactions, an α-proton is abstracted from a carbon adjacent to a carbonyl or carboxyl group by an active site residue, because these protons are acidic by resonance stabilization of the carbanion intermediates. Despite the acidifying effect, the pK a values of the α-protons are typically much higher (∼16–20) than that of an enzymatic base group (<7) involved in the proton abstraction (
      • Gerlt J.A.
      • Gassman P.G.
      ). The unfavorable proton transfer requires the energy given as 2.303RTΔpK a (
      • Gerlt J.A.
      • Gassman P.G.
      ), where ΔpK a is the difference in pK a values between the proton donor and acceptor.
      Δ5-3-Ketosteroid isomerase (KSI)1(EC 5.3.3.1) from Pseudomonas testosteroni catalyzes the isomerization of Δ5- to Δ4-3-ketosteroid by a stereospecific intramolecular transfer of the C4β proton to the C6β position (
      • Eames T.C.
      • Hawkinson D.C.
      • Pollack R.M.
      ,
      • Xue L.
      • Kuliopulos A.
      • Mildvan A.S.
      • Talalay P.
      ,
      • Holman C.M.
      • Benisek W.F.
      ), which is also found in the synthesis of all steroid hormones in mammals. The reaction consists of enolate formation and reketonization that are involved in a wide variety of biologically important reactions of carbonyl compounds. The enzyme, a homodimer in solution, is a “perfect enzyme” enhancing the catalytic rate by a factor of 11 orders of magnitude (
      • Radzicka A.
      • Wolfenden R.
      ). Since the discovery of this enzyme in 1955 (
      • Talalay P.
      • Wang V.S.
      ), it has been intensively studied as a prototype for understanding the chemical and thermodynamic aspects of enzyme-catalyzed C-H bond cleavage. Three residues have been shown by site-directed mutagenesis and kinetic studies to be important for the catalysis: Tyr14 (
      • Kuliopulos A.
      • Talalay P.
      • Mildvan A.S.
      ), Asp38 (
      • Kuliopulos A.
      • Talalay P.
      • Mildvan A.S.
      ), and Asp99(
      • Kim S.W.
      • Cha S.-S.
      • Cho H.-S.
      • Kim J.-S.
      • Ha N.-C.
      • Cho M.-J..J.S.
      • Kim K.-K.
      • Choi K.Y.
      • Oh B.-H.
      ,
      • Wu Z.R.
      • Ebrahimian S.
      • Zawrotny M.E.
      • Thornburg L.D.
      • Perez-Alvarado G.C.
      • Brothers P.
      • Pollack R.M.
      • Summers M.F.
      ). Asp99 is a newly identified catalytic residue on the basis of the solution structure of the enzyme (
      • Wu Z.R.
      • Ebrahimian S.
      • Zawrotny M.E.
      • Thornburg L.D.
      • Perez-Alvarado G.C.
      • Brothers P.
      • Pollack R.M.
      • Summers M.F.
      ) and the crystal structure of a homologous KSI from Pseudomonas putida (
      • Kim S.W.
      • Cha S.-S.
      • Cho H.-S.
      • Kim J.-S.
      • Ha N.-C.
      • Cho M.-J..J.S.
      • Kim K.-K.
      • Choi K.Y.
      • Oh B.-H.
      ). The latter enzyme shares 34% sequence identity with P. testosteroni KSI and retains the three catalytic residues identically. It is generally agreed upon that Asp38 serves as the general base abstracting the C4β proton of the steroid substrate. This proton transfer poses a common major mechanistic enigma of how a strong acid enzymatic group serves as a general base to abstract proton from a much weaker acid group in a substrate. The large disparity in ΔpK a of ∼8 between Asp38 (pK a ∼4.7) (
      • Hawkinson D.C.
      • Pollack R.M.
      • Ambulos Jr., N.P.
      ) and the C4β proton of the steroid substrate (pK a ∼12.7) (
      • Pollack R.M.
      • Mack J.P.G.
      • Eldin S.
      ) requires 11 kcal/mol of energy according to the equation. The energy has to be supplied in the course of the enzyme reaction and/or the ΔpK a should be substantially reduced at the enzyme active site by the transition state stabilization to account for the high turnover rate. It has not yet been settled how Tyr14and Asp99 interact with the dienolic intermediate, and thus the underlying mechanism for the enormous rate enhancement by the enzyme cannot be addressed properly. Two different hydrogen bonding schemes for the transition state stabilization have been proposed. In the first proposal, both Tyr14 and Asp99directly provide hydrogen bonds (H-bonds) to the enolate oxygen of the intermediate (Fig. 1, mechanism I). This has been supported by the model building of the substrate into the solution structure of the free enzyme (
      • Wu Z.R.
      • Ebrahimian S.
      • Zawrotny M.E.
      • Thornburg L.D.
      • Perez-Alvarado G.C.
      • Brothers P.
      • Pollack R.M.
      • Summers M.F.
      ) and by the 2.5 Å crystal structure of P. putida KSI in complex with equilenin (
      • Kim S.W.
      • Cha S.-S.
      • Cho H.-S.
      • Kim J.-S.
      • Ha N.-C.
      • Cho M.-J..J.S.
      • Kim K.-K.
      • Choi K.Y.
      • Oh B.-H.
      ). The second proposal involves a hydrogen-bonded catalytic dyad, Asp99 COOH–Tyr14 OH–O-steroid, consisting of normal hydrogen bonds in the enzyme-substrate complex. As the intermediate dienolate is formed, the H-bond between Asp99and Tyr14 is strengthened to form a low barrier hydrogen bond (LBHB) that facilitates polarization of the C3-keto group of the steroids (Fig. 1, mechanism II). This proposal is primarily based on the detection and assignment to the Asp99 COOH of a highly deshielded proton NMR peak at 18.15 ppm in the presence of dihydroequilenin (
      • Zhao Q.
      • Abeygunawardana C.
      • Gittis A.G.
      • Mildvan A.S.
      ). Such anomalously downfield-shifted resonances have been typically observed for a number of H-bonds between two heteroatoms with an equal pK a in an apolar environment (
      • Gerlt J.A.
      • Gassman P.G.
      ) and attributed to the formation of unusually strong H-bonds (
      • Gerlt J.A.
      • Gassman P.G.
      ,
      • Gerlt J.A.
      • Gassman P.G.
      ,
      • Cleland W.W.
      • Kreevoy M.M.
      ,
      • Frey P.A.
      • Whitt S.A.
      • Tobin J.B.
      ). The resolution (2.5 Å) of the crystal structure ofP. putida KSI complexed with equilenin was criticized as being insufficient to distinguish distances shorter than 0.75 Å and to discern other possible binding modes of equilenin (
      • Massiah M.A.
      • Abeygunawardana C.
      • Gittis A.G.
      • Mildvan A.S.
      ). To resolve this conflicting issue and provide a sound ground for addressing the more significant issue of how the enormous rate enhancement is achieved by the enzyme, we have determined the structure of P. testosteroni KSI in complex with equilenin (Scheme1). The elucidation of the equilenin binding modes observed for the six KSI molecules in the asymmetric unit of the crystal, together with the previously reported structure of the uninhibited enzyme (
      • Cho H.-S.
      • Choi G.
      • Choi K.Y.
      • Oh B.-H.
      ), settles the confusion regarding the catalytic mechanism of this heavily studied enzyme.
      Figure thumbnail gr1
      Figure 1Two different hydorgen bonding schemes proposed for the catalytic mechanism of KSI. In mechanism I, the intermediate is stabilized by one LBHB and one short H-bond. In mechanism II, the intermediate is stabilized by a LBHB from Asp99 to Tyr14, which strengthens the H-bond from Tyr14 to the intermediate. The LBHB is shown bythick dotted lines and a proton being in the middle of the two oxygen atoms.

      REFERENCES

        • Walsh C.
        Enzymatic Reaction Mechanisms. Freeman, New York1984
        • Gerlt J.A.
        • Kozarich J.W.
        • Kenyon G.L.
        • Gassman P.G.
        J. Am. Chem. Soc. 1991; 113: 9667-9669
        • Gerlt J.A.
        • Gassman P.G.
        J. Am. Chem. Soc. 1992; 114: 5928-5934
        • Eames T.C.
        • Hawkinson D.C.
        • Pollack R.M.
        J. Am. Chem. Soc. 1990; 112: 1996-1998
        • Xue L.
        • Kuliopulos A.
        • Mildvan A.S.
        • Talalay P.
        Biochemistry. 1991; 30: 4991-4997
        • Holman C.M.
        • Benisek W.F.
        Biochemistry. 1994; 33: 2672-2681
        • Radzicka A.
        • Wolfenden R.
        Science. 1995; 267: 90-93
        • Talalay P.
        • Wang V.S.
        Biochim. Biophys. Acta. 1955; 18: 300-301
        • Kuliopulos A.
        • Talalay P.
        • Mildvan A.S.
        Biochemistry. 1990; 29: 10271-10280
        • Kim S.W.
        • Cha S.-S.
        • Cho H.-S.
        • Kim J.-S.
        • Ha N.-C.
        • Cho M.-J..J.S.
        • Kim K.-K.
        • Choi K.Y.
        • Oh B.-H.
        Biochemistry. 1997; 36: 14030-14036
        • Wu Z.R.
        • Ebrahimian S.
        • Zawrotny M.E.
        • Thornburg L.D.
        • Perez-Alvarado G.C.
        • Brothers P.
        • Pollack R.M.
        • Summers M.F.
        Science. 1997; 276: 415-418
        • Hawkinson D.C.
        • Pollack R.M.
        • Ambulos Jr., N.P.
        Biochemistry. 1994; 33: 12172-12183
        • Pollack R.M.
        • Mack J.P.G.
        • Eldin S.
        J. Am. Chem. Soc. 1987; 109: 5048-5050
        • Zhao Q.
        • Abeygunawardana C.
        • Gittis A.G.
        • Mildvan A.S.
        Biochemistry. 1997; 36: 14616-14626
        • Gerlt J.A.
        • Gassman P.G.
        J. Am. Chem. Soc. 1993; 115: 11552-11568
        • Gerlt J.A.
        • Gassman P.G.
        Biochemistry. 1993; 32: 11943-11952
        • Cleland W.W.
        • Kreevoy M.M.
        Science. 1994; 264: 1887-1890
        • Frey P.A.
        • Whitt S.A.
        • Tobin J.B.
        Science. 1994; 264: 1927-1930
        • Massiah M.A.
        • Abeygunawardana C.
        • Gittis A.G.
        • Mildvan A.S.
        Biochemistry. 1998; 37: 14701-14712
        • Cho H.-S.
        • Choi G.
        • Choi K.Y.
        • Oh B.-H.
        Biochemistry. 1998; 37: 8325-8330
        • Kim S.W.
        • Choi K.Y.
        J. Bacteriol. 1995; 177: 2602-2605
        • Otwinowski Z.
        • Minor W.
        Methods Enzymol. 1997; 276: 307-326
        • Brünger A.T.
        X-PLOR, Version 3.843. Yale University Press, New Haven, CT1992
        • Platue P.
        • Gueron M.
        J. Am. Chem. Soc. 1982; 104: 7310-7311
        • Frisch M.J.
        Gaussian 94. Gaussian Inc., Pittsburgh, PA1995
        • Lundqvist T.
        • Rice J.
        • Hodge C.N.
        • Basarab G.S.
        • Pierce J.
        • Lindqvist Y.
        Structure. 1994; 2: 937-944
        • Bullock T.L.
        • Clarkson W.D.
        • Kent H.M.
        • Stewart M.
        J. Mol. Biol. 1996; 260: 422-431
        • Kauppi B.
        • Lee K.
        • Carredano E.
        • Parales R.E.
        • Gibson D.T.
        • Eklund H.
        • Ramaswamy S.
        Structure. 1998; 6: 571-586
        • Weintraub H.
        • Alfsen A.
        • Baulieu E.-E.
        Eur. J. Biochem. 1970; 12: 217-221
        • Li Y.K.
        • Kuliopulos A.
        • Mildvan A.S.
        • Talalay P.
        Biochemistry. 1993; 32: 1816-1824
        • Kuliopulos A.
        • Mildvan A.S.
        • Shortle D.
        • Talalay P.
        Biochemistry. 1989; 28: 149-159
        • Zhao Q.
        • Abeygunawardana C.
        • Talalay P.
        • Mildvan A.S.
        Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8220-8224
        • Weintraub H.
        • Vincent F.
        • Baulieu E.-E.
        • Alfsen A.
        Biochemistry. 1977; 16: 5045-5053
        • Bevins C.L.
        • Bantia S.
        • Pollack R.M.
        • Bounds R.L.
        • Kayser R.H.
        J. Am. Chem. Soc. 1984; 106: 4957-4962
        • Luzzati P.V.
        Acta Crystallogr. 1952; 5: 802-810
        • Scheiner S.
        • Kar T.
        J. Am. Chem. Soc. 1995; 117: 6970-6975
        • Shan S.
        • Loh S.
        • Herschlag D.
        Science. 1996; 272: 97-101
        • Ash E.L.
        • Sudmeier J.L.
        • De Fabo E.C.
        • Bachovichin W.W.
        Science. 1997; 278: 1128-1132
        • Cleland W.W.
        • Frey P.A.
        • Gerlt J.A.
        J. Biol. Chem. 1998; 273: 25529-25532
        • Petrounia I.
        • Pollack R.M.
        Biochemistry. 1998; 37: 700-705
        • Jones T.A.
        • Kjeldgaard M.
        O, Version 5.9. Uppsala University, Uppsala, Sweden1993
        • Kraulis P.J.
        J. Appl. Crystallogr. 1991; 24: 946-950