High-resolution crystal structures of delta5-3-ketosteroid isomerase with and without a reaction intermediate analogue

Bacterial Delta5-3-ketosteroid isomerase (KSI) catalyzes a stereospecific isomerization of steroid substrates at an extremely fast rate, overcoming a large disparity of pKa values between a catalytic residue and its target. The crystal structures of KSI from Pseudomonas putida and of the enzyme in complex with equilenin, an analogue of the reaction intermediate, have been determined at 1.9 and 2.5 A resolution, respectively. The structures reveal that the side chains of Tyr14 and Asp99 (a newly identified catalytic residue) form hydrogen bonds directly with the oxyanion of the bound inhibitor in a completely apolar milieu of the active site. No water molecule is found at the active site, and the access of bulk solvent is blocked by a layer of apolar residues. Asp99 is surrounded by six apolar residues, and consequently, its pKa appears to be elevated as high as 9.5 to be consistent with early studies. No interaction was found between the bound inhibitor and the residue 101 (phenylalanine in Pseudomonas testosteroni and methionine in P. putida KSI) which was suggested to contribute significantly to the rate enhancement based on mutational analysis. This observation excludes the residue 101 as a potential catalytic residue and requires that the rate enhancement should be explained solely by Tyr14 and Asp99. Kinetic analyses of Y14F and D99L mutant enzymes demonstrate that Tyr14 contributes much more significantly to the rate enhancement than Asp99. Previous studies and the structural analysis strongly suggest that the low-barrier hydrogen bond of Tyr14 (>7.1 kcal/mol), along with a moderate strength hydrogen bond of Asp99 ( approximately 4 kcal/mol), accounts for the required energy of 11 kcal/mol for the transition-state stabilization.     

Characterization of the three tyrosine residues of delta 5-3-ketosteroid isomerase by time-resolved fluorescence and circular dichroism

delta 5-3-Ketosteroid isomerase (EC 5.3.3.1) of Pseudomonas testosteroni converts delta 5-3-ketosteroids to delta 4-3-ketosteroids via an enolic intermediate. Site-specific mutagenesis has identified Tyr-14 and Asp-38 as the catalytically essential general acid and base, respectively. Three tyrosine residues (Tyr-14, Tyr-55, and Tyr-88) are the only significant fluorophores in the wild-type isomerase. Recent studies of the steady-state fluorescence of the wild-type enzyme and all six mutant enzymes in which one or two tyrosine residues have been mutated to phenylalanine show that the fluorescence intensity of Tyr-14 is very high, that of Tyr-88 is very low, and that of Tyr-55 is intermediate and comparable to that of N-acetyltyrosine amide in solution (Li, Y.-K., Kuliopulos, A., Mildvan, A.S., & Talalay, P. (1993) Biochemistry 32, 1816-1824). Extension of these experiments by time-resolved fluorescence and fluorescence anisotropy measurements demonstrates that Tyr-14, which is in a hydrophobic environment, has an unusually long fluorescence lifetime (4.6 ns) as compared to Tyr-55 (2.0 ns) or Tyr-88 (0.8 ns) and to most protein tyrosine residues (0.2-2 ns). The F?rster distances obtained from the absorption and emission of these tyrosines predict that total quenching of Tyr-14 fluorescence by Tyr-55, and to a lesser degree by Tyr-88, would occur if their orientations were favorable.(ABSTRACT TRUNCATED AT 250 WORDS)     

Active site-directed photoinactivation of delta 5-3-ketosteroid isomerase from Pseudomonas putida dependent on 1,4,6-androstatrien-3-one-17 beta-ol

delta 5-3-Ketosteroid isomerase from Pseudomonas putida is subject to photoinactivation by light of wavelengths greater than 300 nm, specifically in the presence of the competitive inhibitor, 1,4,6-androstatrien-3-one-17 beta-ol (TEO). In the absence of this steroid or in the presence of the nonchromophoric steroidal competitive inhibitor, deoxycholate, the enzyme activity is essentially unaffected by irradiation. Deoxycholate protects the enzyme from the TEO-dependent reaction to a degree which is predictable from the concentrations of deoxycholate and TEO and their respective competitive inhibition constants, thus demonstrating that the inititial velocity of the photoinactivation is dependent upon the fraction of enzyme active sites occupied by TEO. Cholate, which is not a competitive inhibitor, does not protect enzyme activity. Amino acid analyses of hydrochloric acid hydrolysates of the photoinactivated enzyme show no significant differences from that of the native enzyme. However, the fraction of initial enzyme activity remaining correlates quantitatively with the disappearance of one of the four thiol groups in each polypeptide chain of the enzyme. Enzyme irradiated under the same conditions in the absence of TEO does not lose thiol groups.     

Crystal structure and enzyme mechanism of Delta 5-3-ketosteroid isomerase from Pseudomonas testosteroni

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.     

13C NMR relaxation studies of backbone and side chain motion of the catalytic tyrosine residue in free and steroid-bound delta 5-3-ketosteroid isomerase

Side chain and backbone dynamics of the catalytic residue, Tyr-14, in free and steroid-bound delta 5-3-ketosteroid isomerase (EC 5.3.3.1, homodimer, M(r) = 26.8 kDa) have been examined by measurements of longitudinal and transverse 13C relaxation rates and nuclear Overhauser effects at both 500 and 600 MHz (proton frequencies). The data, analyzed using the model-free formalism, yielded an optimized correlation time for overall molecular rotation (tau m) of 17.9 ns, in agreement with the result (18 ns) of fluorescence anisotropy decay measurements [Wu, P., Li, Y.-K., Talalay, P., & Brand, L. (1994) Biochemistry 33, 7415-7422] and Stokes' law calculation (20 ns). The order parameter of the side chain C epsilon of Tyr-14 (S2 = 0.74), which is a measure of the restriction of its high-frequency (nanosecond to picosecond) motion, was significantly lower than that of the backbone C alpha (S2 = 0.82), indicating greater restriction of backbone motion. Upon binding of the steroid ligand, 19-nortestosterone hemisuccinate, a product analog and substrate of the reverse isomerase reaction, S2 of the side chain C epsilon increased from 0.74 to 0.86, while that of the backbone C alpha did not change significantly. Thus, in the steroid complex, the amplitude of high-frequency side chain motion of Tyr-14 became more restricted than that of its backbone which could lower the entropy barrier to catalysis. Lower-frequency (millisecond to microsecond) motion of Tyr-14 at rates comparable to kcat were detected by exchange contributions to transverse relaxation of both C epsilon and C alpha. Steroid binding produced no change in this low-frequency motion of the side chain of Tyr-14, which could contribute to substrate binding and product release, but decreased the exchange contribution to transverse relaxation of the backbone.     

Regulation of messenger ribonucleic acid encoding 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase in the ovine corpus luteum

Three experiments were conducted to determine how steady-state levels of mRNA encoding 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) in the ovine corpus luteum vary 1) between the two steroidogenic luteal cell types, 2) during the estrous cycle, and 3) during prostaglandin F2 alpha (PGF2 alpha)-induced luteolysis. In the first experiment, RNA (10 micrograms) was isolated from purified preparations (n = 4) of large or small steroidogenic luteal cells. Large luteal cells contained 42% more (p < 0.05) message for 3 beta-HSD per microgram RNA than did small luteal cells, while the amount of mRNA for tubulin did not differ between the two types of luteal cells. To determine whether luteal levels of mRNA for 3 beta-HSD differ during the estrous cycle, corpora lutea were collected from cycling ewes (n = 3/day) on Days 3, 6, 9, 12, and 15 postestrus. Levels of mRNA for 3 beta-HSD were similar on Days 3, 6, 9, and 12 but were lower (p < 0.05) on Day 15 postestrus, while levels of mRNA for tubulin were unchanged. In the final experiment, ewes were treated on Day 10 postestrus with two injections of PGF2 alpha (5 mg each) or saline (control) at a 4-h interval. Corpora lutea were collected from ewes (n = 4/treatment) 1 h or 8 h after the second injection of PGF2 alpha or 8 h after the second saline injection.(ABSTRACT TRUNCATED AT 250 WORDS)     

Site-directed mutagenesis of putative active site residues of 5-enolpyruvylshikimate-3-phosphate synthase

The site-directed mutagenesis of a number of proposed active site residues of 5-enolpyruvyl shikimate-3-phosphate (EPSP) synthase is reported. Several of these mutations resulted in complete loss of enzyme activity indicating that these residues are probably involved with catalysis, notably K22R, K411R, D384A, R27A, R100A, and D242A. Of those, K22R, R27A, and D384A did not bind either the substrate shikimate-3-phosphate (S3P) or glyphosate (GLP). The K411R and D242A mutants bind S3P only in the presence of GLP. The kinetic characterization of mutants R100K, K340R, and E418A, which retain activity, is reported. Of those, R100K and K340R do not accumulate enzyme intermediate of enzyme-bound product under equilibrium conditions. These residues, while not essential for catalysis, are most likely important for substrate binding. All of the mutants are shown to be correctly folded by NMR spectroscopy.     

An invariant lysine residue is involved in catalysis at the 3''-5'' exonuclease active site of eukaryotic-type DNA polymerases

The animal health hazards associated with the importation of pork and pork products include four viral agents: foot and mouth disease, classical swine fever (hog cholera), African swine fever, and swine vesicular disease viruses. The safety of importing pork from a zone infected with one or more of these diseases can be adequately determined only through risk assessment. This also applies for the safety of importing pork products which have undergone some form of processing (fully cooked pork products are not counted here). For each disease, the agent (pH and temperature lability), target organs, agent survival in pork and pork products, and agent quantification are discussed. Agent quantification is an input of the risk assessment which measures the viral titres in waste pork and pork products in relation to the oral infective dose estimated for each disease. Two other viral diseases, transmissible gastroenteritis of pigs and porcine reproductive and respiratory syndrome, are presented to illustrate why these two diseases are not hazards when associated with pork and pork products.     

Functional residues at the active site of bovine brain adenosine deaminase

Brain adenosine deaminase was investigated in order to identify amino acid residues essential for its catalytic activity. The pH dependence of log Vmax shows that the enzyme activity depends on two ionizing groups with pK values of 5.4, that must be unprotonated, and 8.4, that must be protonated, for the catalysis. These same groups are observed in the Vmax/Km profiles. The plausible role of histidine residues at the active site of brain adenosine deaminase was proved by chemical modification with (DEP). The histidine specific reagent inactivated the enzyme following a pseudo first-order kinetics with a second-order rate constant of 8.9 10(-3) (+/- 1.8 10(-3)) M-1 min-1. The inhibition of the enzyme with PCMBS was studied monitoring the enzyme activity after incubation with the inhibitor. Brain adenosine deaminase exhibited a characteristic intrinsic tryptophan fluorescence with an emission peak centered at 335 nm. Stern-Volmer quenching parameters in the presence of acrylamide and iodide indicated that tryptophan residues are buried in the native molecule. Tryptophan residues also showed a high heterogeneity that was increased after binding of ground- and transition-state analogs to the enzyme.     

The structure of the MnIIADP-nitrate-lyxose complex at the active site of hexokinase

The coordination scheme of Mn2+ in the hexokinase-MnIIADP-nitrate-lyxose complex has been determined by electron paramagnetic resonance (EPR) spectroscopy with 17O-enriched ligands. Nitrate binds to the active site of hexokinase when MnIIADP and a sugar substrate or analogue are present. The binding of nitrate enhances inhibition by glucose when ADP is present and narrows the EPR signals of the enzyme-bound MnIIADP complex in the presence of sugar substrates or analogues. Experiments using regiospecifically 17O-enriched ADP, 17O-enriched nitrate, and 17O-enriched water establish the coordination scheme of Mn2+. The EPR experiments show that ADP is a beta-monodentate ligand and that nitrate binds directly to Mn2+. Four water molecules complete the coordination sphere of the enzyme-bound Mn2+. The dissociation constant (Kd approximately 8 mM) of nitrate for the complex with enzyme, MnIIADP, and lyxose was obtained from titration experiments. These results suggest that nitrate-stabilized, dead-end complexes of hexokinase may be useful in stabilizing the closed conformation of this "hinge-bending" enzyme for crystallographic experiments.     

Microstructural evolution at the bonding interface during the early-stage infrared active brazing of alumina

Infrared brazing of Al₂O₃ and alloy 42 using a silver-base active braze alloy was investigated at 900 °C for 0 to 300 seconds, with a heating rate of 3000 °C/min. Experimental results show that Ti₃(Cu, Al)₃O intermetallic with various amounts of Al is observed in the reaction layer and plays an important role in the early stage of reactive wetting. A two-layer structure is observed at the reaction interface brazed at 900 °C for 5 seconds. The reaction layer close to the alumina contains large amounts of Al, so the mass balance of the system is maintained. The growth of the reaction layer is not rate controlled by diffusion within the first 120 seconds. After 120 seconds, the rate controlling mechanism of the reaction layer becomes the diffusion control, satisfying the parabolic law. Dynamic wetting angle measurements using a traditional vacuum furnace at the heating rate of 10 °C/min demonstrate that the wetting angle rapidly decreases within the first 150 seconds, especially 0 to 80 seconds, and eventually stabilizes after 600 seconds.