Substrate polarization by residues in Δ⁵‐3‐ketosteroid isomerase probed by site‐directed mutagenesis and UV resonance Raman spectroscopy

Abstract

A5-3-Ketosteroid isomerase (KSI: EC 5.3.3.1) of Pseudomonas testosteroni catalyzes the isomerization of A5-3-ketosteroids to A4-3-ketosteroids by the stereospecific transfer of the steroid 40-proton to the 60-position, using Tyr-14 as a general acid and Asp-38 as a base. Ultraviolet resonance Raman (UVRR) spectra have been obtained for the catalytically active double mutant Y55F + Y88F, which retains Tyr-14 as the only tyrosine residue (referred to as the Y140 mutant), and the Y14F mutant, which has 50,000-fold lower activity. The UVRR results establish that binding of the product analog and competitive inhibitors 19-nortestosterone or 4-fluoro-19-nortestosterone to the Y140 mutant does not result in the formation of deprotonated Tyr-14. The UVRR spectra of the steroid inhibitors show large decreases in the vinyl and carbonyl stretching frequencies on binding to the Y140 enzyme but not on binding to the Y14F enzyme. These changes cannot be mimicked by protonation of the steroids. For 19-nortestosterone, the vinyl and carbonyl stretching frequencies shift down (with respect to the values in aqueous solution) by 18 and 27 cm", respectively, on binding to Y140 KSI. It is proposed that the changes in the steroid resonance Raman spectrum arise from polarization of the enone moiety via the close proximity of the charged Asp-38 side chain to the vinyl group and the directional hydrogen bond between Tyr-14 and the 3-carbonyl oxygen of the steroid enone. The 230-nm-excited UVRR spectra do not, however, show changes that are characteristic of strong hydrogen bonding from the tyrosine hydrogen. It is proposed that this hydrogen bonding is compensated by a second hydrogen bond to the Tyr-14 oxygen from another protein residue.UVRR spectra of the Y14, enzyme obtained using 200 nm excitation show enhancement of the amide I1 and S Raman bands. The secondary structure of KSI was estimated from the amide I1 and S intensities and was found to be low in a-helical structure. The a-helix content was estimated to be in the range of 0-25% (i.e., 10 15%).

Role of the Carbonyl Group in Thioester Chain Length Recognition by the Medium Chain Acyl-CoA Dehydrogenase

“…They presumably help stabilize the partial negative charge developed on the carbonyl oxygen atom in the transition state for substrate dehydrogenation (Gerlt & Gassman, 1993;Engel, 1992;Ghisla et al, 1994;Thorpe & Kim, 1995). Hydrogen-bond formation has been suggested as a significant contributor to the polarization and red-shift of the carbonyl group in keto steroid isomerase (Austin et al, 1992;Hawkinson & Pollack, 1993). In addition, the red-shifts of 20-90 nm observed on binding a variety of cinnamoyl-CoA thioesters to enoyl-CoA hydratase (D'Ordine et al, 1994) are clearly relevant to the present studies.…”

Enzymic and Nonenzymic Polarizations of .alpha.,.beta.-Unsaturated Ketosteroids and Phenolic Steroids. Implications for the Roles of Hydrogen Bonding in the Catalytic Mechanism of .DELTA.5-3-Ketosteroid Isomerase

“…*H*.OTyr-14 after the 4P-proton is removed by Asp-38 and provides an attractive alternative. Further charge delocalization may be achieved by another compensating hydrogen bond donor to Tyr-14, such as a backbone amide NH or a bound water molecule, as suggested by UV resonance Raman studies of the 19-nortestosterone-isomerase complex (Austin et al, 1992(Austin et al, , 1995 and UV spectra of the Y55FTY88F mutant bound to 3-keto~teroids.~ Such a hydrogen bond relay network also allows Tyr-14 to function as either a hydrogen bond donor to the carbonyl group of ketosteroids or in its deprotonated form as a hydrogen bond acceptor that stabilizes the enzyme complex with phenolic steroids (see Figure 1). …”

Insights into the Catalytic Mechanism and Active-Site Environment of Comamonas Testosteroni .DELTA.5-3-Ketosteroid Isomerase as Revealed by Site-Directed Mutagenesis of the Catalytic Base Aspartate-38

“…Uncertainty with respect to the state of protonation of I is indicated by "(H)". matter of some controversy as to whether the intermediate is a dienol or dienolate, with consensus appearing to favor the dienolate (Austin et al, 1992;Zeng et al, 1992;Guthrie & Kluger, 1993;Zhao et al, 1995). Gerlt and Gassman (1993) have recently proposed that the intermediate is neither one, but rather something in between.…”

“…However, the pK, of Y 14 has been directly measured in Y55FlY88F and was found to be 11.6 (Li et al, 1993), suggesting that it is not the titration of Y 14 that is responsible for the observed loss of activity and fluorescence quenching at high pH (Li et al, 1993). Austin et al (1992) have also proposed the existence of a group that donates a hydrogen bond to Tyr-14, on the basis of the results UV resonance Raman spectroscopy studies. They suggested that the most likely candidate for this unidentified functionality would be a lysine residue; however, Mildvan and co-workers have recently singly mutated each of KSIs four lysines and found that all four mutants retain between 43% and 54% of wild-type activity (Li et al, 1993).…”