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.     

Mutational analysis of the three cysteines and active-site aspartic acid 103 of ketosteroid isomerase from Pseudomonas putida biotype B

In order to clarify the roles of three cysteines in ketosteroid isomerase (KSI) from Pseudomonas putida biotype B, each of the cysteine residues has been changed to a serine residue (C69S, C81S, and C97S) by site-directed mutagenesis. All cysteine mutations caused only a slight decrease in the k(cat) value, with no significant change of Km for the substrate. Even modification of the sulfhydryl group with 5,5'-dithiobis(2-nitrobenzoic acid) has almost no effect on enzyme activity. These results demonstrate that none of the cysteines in the KSI from P. putida is critical for catalytic activity, contrary to the previous identification of a cysteine in an active-site-directed photoinactivation study of KSI. Based on the three-dimensional structures of KSIs with and without dienolate intermediate analog equilenin, as determined by X-ray crystallography at high resolution, Asp-103 was found to be located within the range of the hydrogen bond to the equilenin. To assess the role of Asp-103 in catalysis, Asp-103 has been replaced with either asparagine (D103N) or alanine (D103A) by site-directed mutagenesis. For D103A mutant KSI there was a significant decrease in the k(cat) value: the k(cat) of the mutant was 85-fold lower than that of the wild-type enzyme; however, for the D103N mutant, which retained some hydrogen bonding capability, there was a minor decrease in the k(cat) value. These findings support the idea that aspartic acid 103 in the active site is an essential catalytic residue involved in catalysis by hydrogen bonding to the dienolate intermediate.     

Catalytic contribution of phenylalanine-101 of 3-oxo-Delta 5-steroid isomerase

3-Oxo-delta 5-steroid isomerase (KSI, EC 5.3.3.1) from Pseudomonas testosteroni catalyzes the isomerization of a variety of 3-oxo-delta 5-steroids to their conjugated Delta4-isomers through the formation of an intermediate dienolate ion. It has previously been found in our laboratory that the aromatic ring of Phe-101 is important for catalysis. The present work extends these studies. Two double-mutant KSIs (D38E/F101L and D38E/F101A) were prepared to compare the free energy profiles for the reactions catalyzed by these mutants and by D38E. Both double-mutant KSIs show reduced values of kcat at pH 7 compared to D38E ( approximately 25-fold for D38E/F101L and approximately 200-fold for D38E/F101A), similar to the reduced values for F101L and F101A relative to KSI ( approximately 30-fold for F101L and approximately 270-fold for F101A). Free energy profiles for the reactions catalyzed by D38E/F101L and D38E/F101A indicate that the bound transition state(s) and bound intermediate are destabilized when the large aromatic residue Phe-101 in D38E KSI is replaced by the smaller residues Leu or Ala. The pH-rate profiles for D38E, D38E/F101L, and D38E/F101A in the pH range 3.9-8.7 show that the pKa of the catalytic base (Glu-38) is perturbed. In addition, these mutants have significant catalytic activity in the low-pH region.     

Identification of pairwise interactions in the alpha-neurotoxin-nicotinic acetylcholine receptor complex through double mutant cycles

alpha-Neurotoxins are potent inhibitors of the nicotinic acetylcholine receptor (nAChR), binding with high affinity to the two agonist sites located on the extracellular domain. Previous site-directed mutagenesis had identified three residues on the alpha-neurotoxin from Naja mossambica mossambica (Lys27, Arg33, and Lys47) and four residues on the mouse muscle nAChR alpha-subunit (Val188, Tyr190, Pro197, and Asp200) as contributing to binding. In this study, thermodynamic mutant cycle analysis was applied to these sets of residues to identify specific pairwise interactions. Amino acid variants of alpha-neurotoxin from N. mossambica mossambica at position 33 and of the nAChR at position 188 showed strong energetic couplings of 2-3 kcal/mol at both binding sites. Consistently smaller yet significant linkages of 1.6-2.1 kcal/mol were also observed between variants at position 27 on the toxin and position 188 on the receptor. Additionally, toxin residue 27 coupled to the receptor residues 190, 197, and 200 at the alphadelta binding site with observed coupling energies of 1.5-1.9 kcal/mol. No linkages were found between toxin residue Lys47 and the receptor residues studied here. These results provide direct evidence that the two conserved cationic residues Arg33 and Lys27, located on loop II of the toxin structure, are binding in close proximity to the alpha-subunit region between residues 188-200. The toxin residue Arg33 is closer to Val188, where it is likely stabilized by adjacent negative or aromatic residues on the receptor structure. Lys27 is positioned closer to Tyr190, Pro197, and Asp200, where it is likely stabilized through electrostatic interaction with Asp200 and/or cation/pi interactions with Tyr190.     

The responsiveness of generic quality of life instruments in rheumatic diseases. A systematic review of randomized controlled trials

Pseudomonas carboxyl proteinase (PCP), isolated from Pseudomonas sp. 101, is the first example from a prokaryote of unique carboxyl proteinases [EC 3.4.23.33] which are insensitive to aspartic proteinase inhibitors, such as pepstatin, diazoacetyl-DL-norleucine methylester, and 1,2-epoxy-3(p-nitrophenoxy)propane. To identify the catalytic residue(s) of PCP, chemical modification was carried out using carboxyl residue-specific reagents, carbodiimides. PCP was inactivated effectively by N,N'-dicyclohexylcarbodiimide (DCCD) with pseudo-first-order kinetics. For the inactivation, 0.7 mol DCCD was involved per 1 mol PCP. The effects of pH and methanol on the inactivation showed that two carboxyl residues (Asp and/or Glu) were involved in the reaction. The inactivation by DCCD was prevented by a competitive inhibitor, tyrostatin, or a synthetic substrate in a concentration-dependent manner. Based on these data, differential labeling of PCP with DCCD was carried out: Firstly, PCP was treated with cold DCCD in the presence of tyrostatin. After removal of the tyrostatin, which covered the substrate binding site, by dialysis, the PCP was treated with [14C]DCCD to label carboxyl residue(s) essential for its function. Two labeled peptides were isolated by HPLC from a trypsin digest of cold- and [14C]DCCD modified PCP. On analysis of their amino acid sequences, it was revealed that the [14C]DCCD was bound to Asp140 and Glu222 of PCP, respectively. Based on these data, it was strongly suggested that Asp140 and Glu222 of PCP were involved in its catalytic function or substrate binding.     

TIMP-1 contact sites and perturbations of stromelysin 1 mapped by NMR and a paramagnetic surface probe

Surfaces of the 173 residue catalytic domain of human matrix metalloproteinase 3 (MMP-3(DeltaC)) affected by binding of the N-terminal, 126 residue inhibitory domain of human TIMP-1 (N-TIMP-1) have been investigated using an amide-directed, NMR-based approach. The interface was mapped by a novel method that compares amide proton line broadening by paramagnetic Gd-EDTA in the presence and absence of the binding partner. The results are consistent with the X-ray model of the complex of MMP-3(DeltaC) with TIMP-1 (Gomis-Rüth et al. (1997) Nature 389, 77-81). Residues Tyr155, Asn162, Val163, Leu164, His166, Ala167, Ala169, and Phe210 of MMP-3(DeltaC) are protected from broadening by the Gd-EDTA probe by binding to N-TIMP-1. N-TIMP-1-induced exposure of backbone amides of Asp238, Asn240, Gly241, and Ser244 of helix C of MMP-3(DeltaC) to Gd-EDTA confirms that the displacement of the N-terminus of MMP-3(DeltaC) occurs not only in the crystal but also in solution. These results validate comparative paramagnetic surface probing as a means of mapping protein-protein interfaces. Novel N-TIMP-1-dependent changes in hydrogen bonding near the active site of MMP-3(DeltaC) are reported. N-TIMP-1 binding causes the amide of Tyr223 of MMP-3(DeltaC) bound by N-TIMP-1 to exchange with water rapidly, implying a lack of the hydrogen bond observed in the crystal structure. The backbone amide proton of Asn162 becomes protected from rapid exchange upon forming a complex with N-TIMP-1 and could form a hydrogen bond to N-TIMP-1. N-TIMP-1 binding dramatically increases the rate of amide hydrogen exchange of Asp177 of the fifth beta strand of MMP-3(DeltaC), disrupting its otherwise stable hydrogen bond.     

Transcription factor Sp1 is essential for early embryonic development but dispensable for cell growth and differentiation

The three-dimensional structures of the catalytic residue Glu219-->Gln mutant of Pseudomonas stutzeri maltotetraose-forming exo-alpha-amylase, and its complex with carbohydrate obtained by cocrystallization with maltopentaose were determined. Two crystal forms were obtained for the complexed enzyme, and a bound maltotetraose was found in each. The structures were analyzed at 2.2 A and 1.9 A resolution, respectively for the uncomplexed and complexed mutant. These structures were compared with the wild-type enzyme structure. In the complexed crystals, the maltotetraose was firmly bound, extensively interacting with the amino acid environments in the active cleft. The non-reducing end glucose unit was hydrogen bonded to the side-chain of Asp160 and the main-chain nitrogen of Gly158, which seem to be predominantly required for the recognition of the non-reducing end of the substrate that determines the exo-wise degradation of this enzyme. The reducing end glucose unit of bound maltotetraose showed clear deformation, adopting a half-chair conformation with extensive hydrogen bonds to surrounding polypeptides. The C1-atom of this deformed glucose unit lies very close to Asp193OD1 with a distance of 2.6 A. The catalytic residue Asp294 is firmly hydrogen-bonded to the O2 and O3-hydroxyl groups of the deformed reducing end glucose unit. Upon binding of the carbohydrate, small but significant induced fits were observed in the regions of Asp294, Phe156, Ile157, and Asp160. Possible roles of the three catalytic residues are also discussed.     

Potential roles of conserved amino acids in the catalytic domain of the cGMP-binding cGMP-specific phosphodiesterase

The known mammalian 3':5'-cyclic nucleotide phosphodiesterases (PDEs) contain a conserved region located toward the carboxyl terminus, which constitutes a catalytic domain. To identify amino acids that are important for catalysis, we introduced substitutions at 23 conserved residues within the catalytic domain of the cGMP-binding cGMP-specific phosphodiesterase (cGB-PDE; PDE5). Wild-type and mutant proteins were compared with respect to Km for cGMP, kcat, and IC50 for zaprinast. The most dramatic decrease in kcat was seen with H643A and D754A mutants with the decrease in free energy of binding (DeltaDeltaGT) being about 4.5 kcal/mol for each, which is within the range predicted for loss of a hydrogen bond involving a charged residue. His643 and Asp754 are conserved in all known PDEs and are strong candidates to be directly involved in catalysis. Substitutions of His603, His607, His647, Glu672, and Asp714 also produced marked changes in kcat, and these residues are likely to be important for efficient catalysis. The Y602A and E775A mutants exhibited the most dramatic increases in Km for cGMP, with calculated DeltaDeltaGT of 2.9 and 2.8 kcal/mol, respectively, that these two residues are important for cGMP binding in the catalytic site. Zaprinast is a potent competitive inhibitor of cGB-PDE, but the key residues for its binding differ significantly from those that bind cGMP.     

Functional roles of conserved amino acid residues in DNA methyltransferases investigated by site-directed mutagenesis of the EcoRV adenine-N6-methyltransferase

All DNA methyltransferases (MTases) have similar catalytic domains containing nine blocks of conserved amino acid residues. We have investigated by site-directed mutagenesis the function of 17 conserved residues in the EcoRV alpha-adenine-N6-DNA methyltransferase. The structure of this class of MTases has been predicted recently. The variants were characterized with respect to their catalytic activities and their abilities to bind to DNA and the S-adenosylmethionine (AdoMet) cofactor. Amino acids located in motifs X, I, and II are shown to be involved in AdoMet binding (Lys16, Glu37, Phe39, and Asp58). Some of the mutants defective in AdoMet binding are also impaired in DNA binding, suggesting allosteric interactions between the AdoMet and DNA binding site. Asp78 (motif III), which was supposed to form a hydrogen bond to the AdoMet on the basis of the structure predictions, turned out not to be important for AdoMet binding, suggesting that motif III has not been identified correctly. R128A and N130A, having mutations in the putative DNA binding domain, are unable to bind to DNA. Residues located in motifs IV, V, VI, and VIII are involved in catalysis (Asp193, Tyr196, Asp211, Ser229, Trp231, and Tyr258), some of them presumably in binding the flipped target base, because mutations at these residues fail to significantly interfere with DNA and AdoMet binding but strongly reduce catalysis. Our results are in substantial agreement with the structure prediction for EcoRV alpha-adenine-N6-methyltransferase and x-ray structures of other MTases.     

Light and electron microscopic distribution of the AMPA receptor subunit, GluR2, in the spinal cord of control and G86R mutant superoxide dismutase transgenic mice

The NMR structure of the 98 residue beta-elicitin, cryptogein, which induces a defence response in tobacco, was determined using 15N and 13C/15N labelled protein samples. In aqueous solution conditions in the millimolar range, the protein forms a discrete homodimer where the N-terminal helices of each monomer form an interface. The structure was calculated with 1047 intrasubunit and 40 intersubunit NOE derived distance constraints and 236 dihedral angle constraints for each subunit using the molecular dynamics program DYANA. The twenty best conformers were energy-minimized in OPAL to give a root-mean-square deviation to the mean structure of 0.82 A for the backbone atoms and 1.03 A for all heavy atoms. The monomeric structure is nearly identical to the recently derived X-ray crystal structure (backbone rmsd 0.86 A for residues 2 to 97) and shows five helices, a two stranded antiparallel beta-sheet and an omega-loop. Using 1H,15N HSQC spectroscopy the pKa of the N- and C-termini, Tyr12, Asp21, Asp30, Asp72, and Tyr85 were determined and support the proposal of several stabilizing ionic interactions including a salt bridge between Asp21 and Lys62. The hydroxyl hydrogens of Tyr33 and Ser78 are clearly observed indicating that these residues are buried and hydrogen bonded. Two other tyrosines, Tyr47 and Tyr87, show pKa's > 12, however, there is no indication that their hydroxyls are hydrogen bonded. Calculations of theoretical pKa's show general agreement with the experimentally determined values and are similar for both the crystal and solution structures.     

Hydrogen bonding stabilizes globular proteins

It is clear that intramolecular hydrogen bonds are essential to the structure and stability of globular proteins. It is not clear, however, whether they make a net favorable contribution to this stability. Experimental and theoretical studies are at odds over this important question. Measurements of the change in conformational stability, delta (delta G), for the mutation of a hydrogen bonded residue to one incapable of hydrogen bonding suggest a stabilization of 1.0 kcal/mol per hydrogen bond. If the delta (delta G) values are corrected for differences in side-chain hydrophobicity and conformational entropy, then the estimated stabilization becomes 2.2 kcal/mol per hydrogen bond. These and other experimental studies discussed here are consistent and compelling: hydrogen bonding stabilizes globular proteins.     

His...Asp catalytic dyad of ribonuclease A: structure and function of the wild-type, D121N, and D121A enzymes

The side chains of histidine and aspartate residues form a hydrogen bond in the active sites of many enzymes. In serine proteases, the His...Asp hydrogen bond of the catalytic triad is known to contribute greatly to catalysis, perhaps via the formation of a low-barrier hydrogen bond. In bovine pancreatic ribonuclease A (RNase A), the His...Asp dyad is composed of His119 and Asp121. Previously, site-directed mutagenesis was used to show that His119 has a fundamental role, to act as an acid during catalysis of RNA cleavage [Thompson, J. E., and Raines, R. T. (1994) J. Am. Chem. Soc. 116, 5467-5468]. Here, Asp121 was replaced with an asparagine or alanine residue. The crystalline structures of the two variants were determined by X-ray diffraction analysis to a resolution of 1.6 A with an R-factor of 0.18. Replacing Asp121 with an asparagine or alanine residue does not perturb the overall conformation of the enzyme. In the structure of D121N RNase A, Ndelta rather than Odelta of Asn121 faces His119. This alignment in the crystalline state is unlikely to exist in solution because catalysis by the D121N variant is not compromised severely. The steady-state kinetic parameters for catalysis by the wild-type and variant enzymes were determined for the cleavage of uridylyl(3'-->5')adenosine and poly(cytidylic acid), and for the hydrolysis of uridine 2',3'-cyclic phosphate. Replacing Asp121 decreases the values of kcat/Km and kcat for cleavage by 10-fold (D121N) and 10(2)-fold (D121A). Replacing Asp121 also decreases the values of kcat/Km and kcat for hydrolysis by 10(0. 5)-fold (D121N) and 10-fold (D121A) but has no other effect on the pH-rate profiles for hydrolysis. There is no evidence for the formation of a low-barrier hydrogen bond between His119 and either an aspartate or an asparagine residue at position 121. Apparently, the major role of Asp121 is to orient the proper tautomer of His119 for catalysis. Thus, the mere presence of a His...Asp dyad in an enzymic active site is not a mandate for its being crucial in effecting catalysis.     

Quaternary structure sensitive tyrosine residues in human hemoglobin: UV resonance raman studies of mutants at alpha140, beta35, and beta145 tyrosine

Recent studies noted the contribution of alpha42Tyr to the T-R-dependent UV resonance Raman (UVRR) spectral changes of HbA [Nagai, M., et al. (1996) J. Mol. Struct. 379, 65-75; Huang, S., et al. (1997) Biochemistry 36, 6197-6206], but the observed UVRR changes of the Tyr residue cannot be fully interpreted with alpha42Tyr alone. To identify the remaining contributions, the 235 nm-excited UVRR spectra of Tyr mutant Hbs at alpha140, beta35, and beta145 were investigated here. The Fe-His stretching mode demonstrated that all of these mutant Hbs take the T structure in the deoxy form under these experimental conditions. The UVRR change of the Trp residue of these mutants upon the T-R transition was the same as that in HbA, indicating that the T-R-dependent UVRR change of beta37Trp is not due to stacking with Tyr residues but is due to the formation or destruction of a hydrogen bond. The recombinant Hbs beta35Tyr --> Phe and beta35Tyr --> Thr both exhibited UVRR spectra identical with that of HbA, meaning that beta35Tyr is not responsible. In the spectra of des(beta146His,beta145Tyr)Hb with inositol hexaphosphate, the frequency shift of the Tyr RR bands was the same as that in HbA but the intensity enhancement in the CO form was small, suggesting that beta145Tyr contributes to a part of the intensity change, but scarcely relates to the frequency shift. In the spectra of Hb Rouen (alpha140Tyr --> His), the frequency shifts of bands at 1617 (Y8a) and 1177 (Y9a) cm-1 following ligation were half of those in HbA, while the intensity enhancement was not detected. This result means that alpha140Tyr is responsible for both the frequency shift and the intensity changes. It is suggested that the frequency shift of the Tyr RR bands upon the T --> R transition is due to changes in the hydrogen bonding state of alpha42- and alpha140Tyr and that the intensity enhancement is due to changes in the environment of the penultimate Tyr in both alpha and beta subunits (alpha140 and beta145). These alterations in the vibrational spectra clearly demonstrate which tyrosine residues are involved in the T-R transition as a result of modification of their local environments.     

Contribution of hydrogen bonds to the conformational stability of human lysozyme: calorimetry and X-ray analysis of six tyrosine --> phenylalanine mutants

The contribution of hydrogen bonds to the conformational stability of human lysozyme was investigated by the combination of calorimetric and X-ray analyses of six Tyr --> Phe mutants. Unfolding Delta G and unfolding Delta H values of the Tyr --> Phe mutant proteins were changed by from +0.3 to -4.0 kJ/mol and from 0 to -16 kJ/mol, respectively, compared to those of the wild-type protein. The net contribution of a hydrogen bond at a specific site to stability (Delta Gwild/HB), considering factors affected by substitutions, was evaluated on the basis of X-ray structures of the mutant proteins. In the present study, one of six mutant proteins was suitable for evaluating the strength of the hydrogen bond. Delta Gwild/HB for the intramolecular hydrogen bond at Tyr124 was evaluated to be 7.5 kJ/mol. Results of the analysis of other mutants also suggest that hydrogen bonds of the hydroxyl group of Tyr, including the hydrogen bond with a water molecule, contribute to the stabilization of the human lysozyme.     

Free-energy simulations of the retinal cis --> trans isomerization in bacteriorhodopsin

Free-energy profiles for ground-state cis --> trans isomerization of retinal in vacuum, in solution, and in the protein bacteriorhodopsin are calculated using free-energy simulations. The free-energy barriers in the protein were 9 kcal/mol for ionized Asp85 and 14 kcal/mol for neutral Asp85, significantly lower than those found in solution (18 kcal/mol) or vacuum (19 kcal/mol). Therefore, bacteriorhodopsin can be said to act as a catalyst in the isomerization. The barrier in the protein is due mainly to stabilization of the transition state through favorable nonbonded interactions with the protein part of the system, with internal strain and interactions with solvent playing minor roles. The protonated Asp85 simulation models the behavior of the system in the N --> O transition. Our calculated 14 kcal/mol barrier and 4-ms relaxation time for this process are in excellent agreement with experimentally measured values of 12 kcal/mol and 5 ms, respectively. The ionized Asp85 simulation models two hypothetical processes: the N --> O transition with a proton removed from Asp85 and the initial BR568 --> L transition on the ground-state energy surface. The cis-trans isomerization barrier in this system is 9 kcal/mol, the lowest of all the studied cases. The presence of the charged carboxylate group in the ionized Asp85 system leads to strong stabilization of the transition state by interactions with the surroundings and changes the distance between Asp85 and the Schiff base proton compared to the corresponding distance in the neutral Asp85 system. This suggests that the protonation of Asp85 plays an important role in regulating access to the Schiff base proton. For both Asp85 ionization states the calculated cis-trans free-energy difference was close to 0, indicating that the protein can accommodate both retinal isomers equally well. The computed negligible difference between the N and O free-energy levels is in accord with experimental data.     

Fourier transform infrared evidence against Asp beta 99 protonation in hemoglobin: nature of the Tyr alpha 42-Asp beta 99 quaternary H-bond

The Tyr alpha 42-Asp beta 99 intersubunit H-bond stabilizes the T quaternary structure in hemoglobin (Hb) tetramers. We had proposed that Tyr alpha 42 acts as an acceptor in this H-bond, because the tyrosine Y8a/8b and Y7a' UVRR (ultraviolet resonance Raman) bands shift in directions opposite to those expected if tyrosine is an H-bond donor. If Asp beta 99 is the H-bond donor, then it must be protonated in the T state, and would be a previously unrecognized contributor to the Bohr effect. This implication was strengthened by the discovery that an R-minus-T difference FTIR (Fourier transform infrared) band at 1693 cm-1, which might be a signal from protonated carboxylate, is missing in Hb Kempsey, a mutant in which Asp beta 99 is replaced by Asn. However, we now find that this FTIR signal is insensitive to 13C-labeling of the aspartate residues in Hb, and cannot arise from protonated Asp beta 99. There are no other difference signals in the 1700 cm-1 region at a sensitivity of one COOH group. We conclude that Asp beta 99 is not protonated, and that the anomalous UVRR shifts must arise from compensating polarization of the Tyr alpha 42 OH. Candidates for this compensation are the H-bond donated by the Asp beta 94 backbone NH, and the nearby positive charge of Arg beta 40.     

p53 protein expression in breast cancer as related to histopathological characteristics and prognosis

Reaction of Klebsiella aerogenes urease with diethylpyrocarbonate (DEP) led to a pseudo-first-order loss of enzyme activity by a reaction that exhibited saturation kinetics. The rate of urease inactivation by DEP decreased in the presence of active site ligands (urea, phosphate, and boric acid), consistent with the essential reactive residue being located proximal to the catalytic center. The pH dependence for the rate of inactivation indicated that the reactive residue possessed a pKa of 6.5, identical to that of a group that must be deprotonated for catalysis. Full activity was restored when the inactivated enzyme was treated with hydroxylamine, compatible with histidinyl or tyrosinyl reactivity. Spectrophotometric studies were consistent with DEP derivatization of 12 mol of histidine/mol of native enzyme. In the presence of active site ligands, however, approximately 4 mol of histidine/mol of protein were protected from reaction. Each protein molecule is known to possess two catalytic units; hence, we propose that urease possesses at least one essential histidine per catalytic unit.     

The 0.78 A structure of a serine protease: Bacillus lentus subtilisin

Ultrahigh-resolution X-ray diffraction data from cryo-cooled, B. lentus subtilisin crystals has been collected to a resolution of 0.78 A. The refined model coordinates have a rms deviation of 0.22 A relative to the same structure determined at room temperature and 2.0 A resolution. Several regions of main-chain and side-chain disorder have been identified for 21 out of 269 residues in one polypeptide chain. Hydrogen atoms appear as significant peaks in the Fo - Fc difference electron density map, and carbon, nitrogen, and oxygen atoms can be differentiated. The estimated standard deviation (ESD) for all main-chain non-hydrogen bond lengths is 0.009 A and 0.5 degrees for bond angles based on an unrestrained full-matrix least-squares refinement. Hydrogen bonds are resolved in the serine protease catalytic triad (Ser-His-Asp). Electron density is observed for an unusual, short hydrogen bond between aspartic acid and histidine in the catalytic triad. The hydrogen atom, identified by NMR in numerous serine proteases, appears to be shared by the heteroatoms in the bond. This represents the first reported correlation between detailed chemical features identified by NMR and those in a cryo-cooled crystallographic structure determination at ultrahigh resolution. The short hydrogen bond, designated "catalytic hydrogen bond", occurs as part of an elaborate hydrogen bond network, involving Asp of the catalytic triad. While unusual, these features appear to have conserved analogues in other serine protease families although specific details differ from family to family.     

Crystal structures of nucleic acid complexes of ribonuclease U2

Crystal structures of adenine-specific Ustilago sphaerogena ribonuclease U2 complexed with the substrate analogues, d(ApG), d(ApGpG), and d(ApGpC), with the intermediate analogue, 2',3'-O-isopropylidene-adenosine, and with the product, 3'-AMP, have been determined. In each structure, the adenine base is recognized by the enzyme with four hydrogen-bonds. In the substrate analogue structures, the second base of guanine is sandwiched between His 101 and Tyr 107 side-chains, and forms two hydrogen-bonds with Tyr 107 O and Asp 108 O delta 1 atoms. The third base of the trinucleotides is in van der Waals interaction with the Tyr 78 side-chain. The phosphate group between the second and third nucleosides forms two hydrogen-bonds with the side chains of Asp 37 and Tyr 78. Oxygen atoms of the scissile phosphate group are involved in interactions with catalytic residues of Tyr 39, His 41, Glu 62, Arg 85, and His 101. These interactions indicate that either His 41 or Glu 62 acts as a general base and His 101 acts as a general acid in the first step of RNA hydrolysis.     

Properties of the His57-Asp102 dyad of rat trypsin D189S in the zymogen, activated enzyme, and alpha1-proteinase inhibitor complexed forms

Structural and biochemical studies suggest that serpins induce structural rearrangements in their target serine-proteinases. Previous NMR studies of the complex between a serpin, alpha1-proteinase inhibitor, and a mutant of recombinant rat trypsin (the Asp189 to Ser mutant, D189S, which is much more stable than wild-type rat trypsin against autoproteolysis) provided information about the state of catalytic residues in this complex: the hydrogen bond between Asp102 and His57 remains intact in the complex, and spectral properties of His57 are more like those of the zymogen than of the activated enzyme (G. Kaslik, et al., 1997, Biochemistry 36, 5455-5464). Here we report the protonation and exchange behavior of His57 of recombinant rat trypsin D189S in three states: the zymogen, the active enzyme, and the complex with human alpha1-proteinase inhibitor and compare these with analogous behavior of His57 of bovine chymotrypsinogen and alpha-chymotrypsin. In these studies the pKa of His57 has been determined from the pH dependence of the 1H NMR signal from the Hdelta1 proton of histidine in the Asp102-His57 dyad, and a measure of the accessibility of this part of the active site has been obtained from the rate of appearance of this signal following its selective saturation. The activation of rat trypsinogen D189S (zymogen, pKa = 7.8 +/- 0.1; Hill coefficient = 0. 86 +/- 0.05) decreased the pKa of His57 by 1.1 unit and made the protonation process cooperative (active enzyme, pKa = 6.7 +/- 0.1; Hill coefficient = 1.37 +/- 0.08). The binding of alpha1-proteinase inhibitor to trypsin D189S led to an increase in the pKa value of His57 to a value higher than that of the zymogen and led to negative cooperativity in the protonation process (complex, pKa = 8.1 +/- 0. 1; Hill coefficient = 0.70 +/- 0.08), as was observed for the zymogen. In spite of these differences in the pKa of His57 in the zymogen, active enzyme, and alpha1-proteinase inhibitor complex, the solvent exchange lifetime of the His57 Hdelta1 proton was the same, within experimental error, in all three states (lifetime = 2 to 12.5 ms). The linewidth of the 1H NMR signal from the Hdelta1 proton of His57 was relatively sharp, at temperatures between 5 and 20 degrees C at both low pH (5.2) and high pH (10.0), in spectra of bovine alpha-chymotrypsin, recombinant rat trypsin D189S, and the complex between rat trypsin D189S and human alpha1-proteinase inhibitor; however, in spectra of the complex between alpha-chymotrypsin and human alpha1-proteinase inhibitor, the peak was broader and could be well-resolved only at the lower temperature (5 degrees C).