⌬ 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...

The infectious agent of transmissible spongiform encephalopathies (TSE) is believed to comprise, at least in part, the prion protein (PrP). Other molecules can modulate the conversion of the normal PrP(C) into the pathological conformer (PrP(Sc)), but the identity and mechanisms of action of the key physiological factors remain unclear. PrP can bind to nucleic acids with relatively high affinity. Here, we report small-angle X-ray scattering (SAXS) and nuclear magnetic resonance spectroscopy measurements of the tight complex of PrP with an 18 bp DNA sequence. This double-stranded DNA sequence (E2DBS) binds with nanomolar affinity to the full-length recombinant mouse PrP. The SAXS data show that formation of the rPrP-DNA complex leads to larger values of the maximum dimension and radius of gyration. In addition, the SAXS studies reveal that the globular domain of PrP participates importantly in the formation of the complex. The changes in NMR HSQC spectra were clustered in two major regions: one in the disordered portion of the PrP and the other in the globular domain. Although interaction is mediated mainly through the PrP globular domain, the unstructured region is also recruited to the complex. This visualization of the complex provides insight into how oligonucleotides bind to PrP and opens new avenues to the design of compounds against prion diseases.

Bacterial Delta 5-3-ketosteroid isomerase (KSI) from Pseudomonas testosteroni has been intensively studied as a prototype for understanding an enzyme-catalyzed allylic rearrangement involving intramolecular proton transfer. Asp38 serves as a general base to abstract the proton from the steroid C4-H, which is a much stronger base than the carboxyl group of this residue. This unfavorable proton transfer requires 11 kcal/mol of energy which has to be provided by favorable interactions between catalytic residues and substrate in the course of the catalytic reaction. How this energy is provided at the active site of KSI has been a controversial issue, and inevitably the enzyme mechanism is not settled. To resolve these issues, we have determined the crystal structure of this enzyme at 2.3 A resolution. The crystal structure revealed that the active site environment of P. testosteroni KSI is nearly identical to that of Pseudomonas putida KSI, whose structure in complex with a reaction intermediate analogue we have determined recently. Comparison of the two structures clearly indicates that the two KSIs should share the same enzyme mechanism involving the stabilization of the dienolate intermediate by the two direct hydrogen bonds to the dienolate oxyanion, one from Tyr14 OH and the other from Asp99 COOH. Mutational analysis of the two residues and other biochemical data strongly suggest that the hydrogen bond of Tyr14 provides the more significant contribution than that of Asp99 to the requisite 11 kcal/mol of energy for the catalytic power of KSI.

Delta(5)-3-Ketosteroid isomerase from Pseudomonas putida biotype B is one of the most proficient enzymes catalyzing an allylic isomerization reaction at rates comparable to the diffusion limit. The hydrogen-bond network (Asp99... Wat504...Tyr14...Tyr55...Tyr30) which links the two catalytic residues, Tyr14 and Asp99, to Tyr30, Tyr55, and a water molecule in the highly apolar active site has been characterized in an effort to identify its roles in function and stability. The DeltaG(U)(H2O) determined from equilibrium unfolding experiments reveals that the elimination of the hydroxyl group of Tyr14 or Tyr55 or the replacement of Asp99 with leucine results in a loss of conformational stability of 3.5-4.4 kcal/mol, suggesting that the hydrogen bonds of Tyr14, Tyr55, and Asp99 contribute significantly to stability. While decreasing the stability by about 6.5-7.9 kcal/mol, the Y55F/D99L or Y30F/D99L double mutation also reduced activity significantly, exhibiting a synergistic effect on k(cat) relative to the respective single mutations. These results indicate that the hydrogen-bond network is important for both stability and function. Additionally, they suggest that Tyr14 cannot function efficiently alone without additional support from the hydrogen bonds of Tyr55 and Asp99. The crystal structure of Y55F as determined at 1.9 A resolution shows that Tyr14 OH undergoes an alteration in orientation to form a new hydrogen bond with Tyr30. This observation supports the role of Tyr55 OH in positioning Tyr14 properly to optimize the hydrogen bond between Tyr14 and C3-O of the steroid substrate. No significant structural changes were observed in the crystal structures of Y30F and Y30F/Y55F, which allowed us to estimate approximately the interaction energies mediated by the hydrogen bonds Tyr30...Tyr55 and Tyr14...Tyr55. Taken together, our results demonstrate that the hydrogen-bond network provides the structural support that is needed for the enzyme to maintain the active-site geometry optimized for both function and stability.