Kinetic and Ultraviolet Spectroscopic Studies of Active-Site Mutants of Δ5-3-Ketosteroid Isomerase

Athan Kuliopulos, Albert S. Mildvan, David Shortle, Paul Talalay

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Δ5-3-Ketosteroid isomerase (EC of Pseudomonas testosteroni promotes the highly efficient isomerization of Δ5-3-ketosteroids to Δ4-3-ketosteroids by means of a direct and stereospecific transfer of the 4β-proton to the 6β-position, via an enolic intermediate. An acidic residue responsible for the protonation of the 3-carbonyl function of the steroid and a basic group concerned with the proton transfer have been implicated in the catalytic mechanism. Recent NMR studies with a nitroxide spin-labeled substrate analogue have allowed positioning of the steroid into the 2.5-Å X-ray crystal structure of the enzyme [Kuliopulos, A., Westbrook, E. M., Talalay, P., & Mildvan, A. S. (1987) Biochemistry 26, 3927-3937], thereby corroborating the approximate location of the steroid binding site deduced from a difference Fourier X-ray diffraction map of the 4-(acetoxymercuri)estradiol-isomerase complex [Westbrook, E. M., Piro, O. E., & Sigler, P. B. (1984) J. Biol. Chem. 259, 9096-9103], The steroid lies in a hydrophobic cavity near Asp-38, Tyr-14, and Tyr-55. In order to assess the role of these amino acid residues in catalysis, the gene for isomerase was cloned, sequenced, and overexpressed in Escherichia coli [Kuliopulos, A., Shortle, D., & Talalay, P. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8893-8897], and the following mutants were prepared: Asp-38 to asparagine (D38N) and Tyr-14 and Tyr-55 to phenylalanine (Y14F and Y55F, respectively). The kcat value of the D38N mutant enzyme is 105.6-fold lower than that of the wild-type enzyme, suggesting that Asp-38 functions as the base which abstracts the 4β-proton of the steroid in the rate-limiting step. Threefold lower Km values in all mutants indicate tighter binding of the substrate to the more hydrophobic sites. In comparison with the wild-type enzyme, the Y55F mutant shows only a 4-fold decrease in kcat while the YMF mutant shows a 104.7-fold decrease in kcat, suggesting that Tyr-14 is the general acid. The red shift of the ultraviolet absorption maximum of the competitive inhibitor 19-nortestosterone from 248 to 258-260 nm, which occurs upon binding to the wild-type enzyme [Wang, S. F., Kawahara, F. S., & Talalay, P. (1963) J. Biol. Chem. 238, 576-585], is mimicked in strong acid. This spectral shift was also observed with the D38N and Y55F mutants, but not on binding of the steroid to the Y14F mutant. These findings provide further evidence that the phenolic hydroxyl group of Tyr-14 is essential for protonation of the 3-carbonyl group of the steroid. Upon binding of 17β-estradiol to the wild-type enzyme and the D38N and Y55F mutants, the ultraviolet absorption spectrum of the steroid undergoes profound changes that resemble those observed upon ionization of the phenolic hydroxyl group in base. In contrast, binding of 17β-estradiol to the Y14F mutant does not affect the spectrum of the steroid, indicating that Tyr-14 is probably also required for deprotonation of the 3-hydroxyl group of the enolic steroid. From computer-modeling studies of substrate docked into the 2.5-Å X-ray crystal structure of the enzyme, we conclude that Asp-38 and Tyr-14, after a small change in orientation of the latter, are optimally positioned for a stereoelectronically favorable, antarafacial, enolization of the ketosteroid substrate in the rate-limiting first half of the isomerization reaction.

Original languageEnglish (US)
Pages (from-to)149-159
Number of pages11
Issue number1
StatePublished - 1989

ASJC Scopus subject areas

  • Biochemistry


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