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

Abstract

⌬ 5 -3-Ketosteroid isomerase from Pseudomonas testosteroni has been intensively studied as a prototype to understand an enzyme-catalyzed allylic isomerization. Asp 38 (pK a ϳ4.7) was identified as the general base abstracting the steroid C4␤ proton (pK a ϳ12.7) 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 Tyr 14 OH and Asp 99 COOH provide direct hydrogen bonds to the oxyanion of equilenin. The result negates the catalytic dyad mechanism in which Asp 99 donates the hydrogen bond to Tyr 14 , 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 Tyr 14 OH forms a low barrier hydrogen bond with the dienolic oxyanion of the intermediate.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 (1, 2). In almost all the reactions, an ␣-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 ␣-protons are typically much higher (ϳ16 -20) than that of an enzymatic base group (Ͻ7) involved in the proton abstraction (3). The unfavorable proton transfer requires the energy given as 2.303RT⌬pK a (3), where ⌬pK a is the difference in pK a values between the proton donor and acceptor.⌬ 5 -3-Ketosteroid isomerase (KSI) 1 (EC 5.3.3.1) from Pseudomonas testosteroni catalyzes the isomerization of ⌬ 5 -to ⌬ 4 -3-ketosteroid by a stereospecific intramolecular transfer of the C4␤ proton to the C6␤ position (4 -6), 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 (7). Since the discovery of this enzyme in 1955 (8), 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: Tyr 14 (9), Asp 38 (9), and Asp 99 (10, 11). Asp 99 is a newly identified cataly...

Detection of Large pK Perturbations of an Inhibitor and a Catalytic Group at an Enzyme Active Site, a Mechanistic Basis for Catalytic Power of Many Enzymes

“…carrying out 1 H NMR experiments on the D38N-, D38N/D99L-, and D38N/Y14F-PI mutants in the presence of equilenin, we previously assigned the 16.8 ppm resonance to the LBHB between the Tyr 14 OH and the C-3 oxyanion of equilenin (15). The H-bond is relatively short (2.57 Å, Fig.…”

“…The most unambiguous identification of LBHB was claimed to be the detection of the unusual NMR chemical shift for a participating proton (9). In aqueous solution, characteristically deshielded NMR resonances have been observed for several enzymes (12)(13)(14)(15). Among them, the bacterial KSIs are known to exhibit such NMR signals emanating from enzyme-inhibitor interactions.…”

“…Although the chemical nature of catalytic mechanisms is well understood for many enzymes, how enzymes are able to accelerate reaction rate by 10 8 -10 15 has remained an entirely unresolved issue. For example, the energetics of the seemingly simple heterolytic C-H bond cleavage is enigmatic, although it is a fundamental process found in a wide variety of biological reactions such as racemization, transamination, and isomerization (1,2).…”

Origin of the Different pH Activity Profile in Two Homologous Ketosteroid Isomerases

“…The pK a values of catalytic residues can be affected by the local microenvironment of the active site in the enzyme (18). The environment of Asp-38 in TI-WT is very similar to that in PI-WT as judged by crystallographic data of both wild-type enzymes (11,13,19). However, there is a significant difference in that the carboxyl oxygen of Asp-38 is hydrogen-bonded to NH on the indole ring of Trp-116 only in PI-WT (Fig.…”

Novel Approach for Identifying Key Residues in Enzymatic Reactions: Proton Abstraction in Ketosteroid Isomerase

“…proton transfer from a substrate to an Asp. The catalytic reaction and the active site structure of KSI have been studied extensively by experimental techniques [12][13][14][15][16][17][18][19][20][21] and by theoretical computations [22][23][24][25][26][27][28] . It has been shown that KSI works by a preorganized active site which electrostatically stabilizes the intermediate state and the transition states via hydrogen bonds to the substrate.…”