Hydroxyl Group Of Tyr Research Articles

Role of active site tyrosines in dynamic aspects of DNA binding by AP endonuclease

AP endonuclease (AP endo), a key enzyme in repair of abasic sites in DNA, makes a single nick 5′ to the phosphodeoxyribose of an abasic site (AP-site). We recently proposed a novel mechanism, whereby the enzyme uses a key tyrosine (Tyr 171) to directly attack the scissile phosphate of the AP-site. We showed that loss of the tyrosyl hydroxyl from Tyr 171 resulted in dramatic diminution in enzymatic efficiency. Here we extend the previous work to compare binding/recognition of AP endo to oligomeric DNA with and without an AP-site by wild type enzyme and several tyrosine mutants including Tyr 128, Tyr 171 and Tyr 269. We used single turnover and electrophoretic mobility shift assays. As expected, binding to DNA with an AP-site is more efficient than binding to DNA without one. Unlike catalytic cleavage by AP endo, which requires both hydroxyl and aromatic moieties of Tyr 171, the ability to bind DNA efficiently without an AP-site is independent of an aromatic moiety at position 171. However, the ability to discriminate efficiently between DNA with and without an AP-site requires tyrosine at position 171. Thus, AP endo requires a tyrosine at the active site for the properties that enable it to behave as an efficient, processive endonuclease.

Murine Thrombin Lacks Na+ Activation but Retains High Catalytic Activity

Human thrombin utilizes Na+ as a driving force for the cleavage of substrates mediating its procoagulant, prothrombotic, and signaling functions. Murine thrombin has Asp-222 in the Na+ binding site of the human enzyme replaced by Lys. The charge reversal substitution abrogates Na+ activation, which is partially restored with the K222D mutation, and ensures high activity even in the absence of Na+. This property makes the murine enzyme more resistant to the effect of mutations that destabilize Na+ binding and shift thrombin to its anticoagulant slow form. Compared with the human enzyme, murine thrombin cleaves fibrinogen and protein C with similar k(cat)/K(m) values but activates PAR1 and PAR4 with k(cat)/K(m) values 4- and 26-fold higher, respectively. The significantly higher specificity constant toward PAR4 accounts for the dominant role of this receptor in platelet activation in the mouse. Murine thrombin can also cleave substrates carrying Phe at P1, which potentially broadens the repertoire of molecular targets available to the enzyme in vivo.

Hydrogen Bonding in Human Manganese Superoxide Dismutase Containing 3-Fluorotyrosine

Incorporation of 3-fluorotyrosine and site-specific mutagenesis has been utilized with Fourier transform infrared (FTIR) spectroscopy and x-ray crystallography to elucidate active-site structure and the role of an active-site residue Tyr 34 in human manganese superoxide dismutase (MnSOD). Calculated harmonic frequencies at the B3LYP/6-31G** level of theory for L-tyrosine and its 3-fluorine substituted analog are compared to experimental frequencies for vibrational mode assignments. Each of the nine tyrosine residues in each of the four subunits of the homotetramer of human MnSOD was replaced with 3-fluorotyrosine. The crystal structures of the unfluorinated and fluorinated wild-type MnSOD are nearly superimposable with the root mean-square deviation for 198 α-carbon atoms at 0.3 Å. The FTIR data show distinct vibrational modes arising from 3-fluorotyrosine in MnSOD. Comparison of spectra for wild-type and Y34F MnSOD showed that the phenolic hydroxyl of Tyr 34 is hydrogen bonded, acting as a proton donor in the active site. Comparison with crystal structures demonstrates that the hydroxyl of Tyr 34 is a hydrogen bond donor to an adjacent water molecule; this confirms the participation of Tyr 34 in a network of residues and water molecules that extends from the active site to the adjacent subunit.

Tyrosine-612 in PDE5 contributes to higher affinity for vardenafil over sildenafil

Despite close structural similarity, vardenafil (Levitra) is 32-fold more potent than sildenafil (Viagra) to inhibit cGMP-binding cGMP-specific PDE (PDE5); this is due to differences between their heterocyclic rings. In co-crystals with PDE5, one of the rings of vardenafil or sildenafil interacts with Tyr(612), a catalytic site AA, via (1) a hydrogen bond with a water molecule and (2) hydrophobic bonds. For mutant PDE5(Y612F), which ablates hydrogen-bonding potential, vardenafil or sildenafil inhibition was strengthened (2.2- or 3.0-fold, respectively), implying that the Tyr(612) hydroxyl is a negative determinant for these inhibitors. For mutant PDE5(Y612A), which ablates both hydrogen bonding and hydrophobic-bonding potential, vardenafil inhibition was weakened much more than sildenafil inhibition (122- and 26-fold, respectively), suggesting that hydrophobic bonds involving Tyr(612) are stronger for vardenafil than for sildenafil.

A Single Aromatic Amino Acid at the Carboxyl Terminus of Helicobacter pylori α1,3/4 Fucosyltransferase Determines Substrate Specificity

Fucosyltransferases (FucT) from different Helicobacter pylori strains display distinct Type I (Galbeta1,3GlcNAc) or Type II (Galbeta1,4GlcNAc) substrate specificity. FucT from strain UA948 can transfer fucose to the OH-3 of Type II acceptors as well as to the OH-4 of Type I acceptors on the GlcNAc moiety, so it has both alpha1,3 and alpha1,4 activities. In contrast, FucT from strain NCTC11639 has exclusive alpha1,3 activity. Our domain swapping study (Ma, B., Wang, G., Palcic, M. M., Hazes, B., and Taylor, D. E. (2003) J. Biol. Chem. 278, 21893-21900) demonstrated that exchange of the hypervariable loops, (347)DNPFIFC(353) in 11639FucT and (345)CNDAHYSALH(354) in UA948FucT, were sufficient to either confer or abolish alpha1,4 activity. Here we performed alanine scanning site-directed mutagenesis to identify which amino acids within (345)CNDAHYSALH(354) of UA948FucT confer Type I substrate specificity. The Tyr(350) --> Ala mutation dramatically reduced alpha1,4 activity without lowering alpha1,3 activity. None of the other alanine substitutions selectively eliminated alpha1,4 activity. To elucidate how Tyr(350) determines alpha1,4 specificity, mutants Tyr(350) --> Phe, Tyr(350) --> Trp, and Tyr(350) --> Gly were constructed in UA948FucT. These mutations did not decrease alpha1,3 activity but reduced the alpha1,4 activity to 66.9, 55.6, and 3.1% [corrected] of wild type level, respectively. Apparently the aromatic nature, but not the hydroxyl group of Tyr(350), is essential for alpha1,4 activity. Our data demonstrate that a single amino acid (Tyr(350)) in the C-terminal hypervariable region of UA948FucT determines Type I acceptor specificity. Notably, a single aromatic residue (Trp) has also been implicated in controlling Type I acceptor preference for human FucT III, but it is located in an N-terminal hypervariable stem domain.

Mechanistic Roles of Ser-114, Tyr-155, and Lys-159 in 3α-Hydroxysteroid Dehydrogenase/Carbonyl Reductase from Comamonas testosteroni

3alpha-hydroxysteroid dehydrogenase/carbonyl reductase (3alpha-HSD/CR) from Comamonas testosteroni, a short chain dehydrogenase/reductase, catalyzes the oxidation of androsterone with NAD+ to form androstanedione and NADH. A catalytic triad of Ser-114, Tyr-155, and Lys-159 in 3alpha-HSD/CR has been proposed based on structural analysis and sequence alignment of the short chain dehydrogenase/reductase family. The 3alpha-HSD/CR-catalyzed reaction has not been kinetically analyzed in detail, however. In this study, we combined steady-state kinetics, site-directed mutagenesis, and pH profile to explore the function of Ser-114, Tyr-155, and Lys-159 in 3alpha-HSD/CR-catalyzed reaction. The catalytic efficiency of wild-type and mutants S114A, Y155F, K159A, and Y155F/K159A is 4.3 x 10(7), 7.3 x 10(4), 1.7 x 10(4), 2.4 x 10(5), and 71 m(-1)s(-1), respectively. The values of pKa on kcat/Km for the wild-type, S114A, Y155F, K159A, and Y155F/K159A are 7.2, 7.4, 8.4, 9.1, and 10.2, respectively. Mutant S114A/Y155F exhibits a pH-independent profile with 10(-5) times of wild-type activity at pH 10.5. The activity decreases as the pH lowers, which indicates that a functional group with an apparent pKa of 7.2 is involved in the general base catalysis for wild-type 3alpha-HSD/CR. The pKa shift to 9.1 for mutant K159A suggests the role of Lys-159 is to lower the pKa of the residues involved in the general base catalysis. Because pH dependence is observed for both S114A and Y155F mutants and pH independence is observed in S114A/Y155F, Tyr-155 may be important as a general base catalysis in the wild-type, whereas Ser-114 may act as a general base on mutant Y155F to catalyze the reaction.

Tyrosine 547 Constitutes an Essential Part of the Catalytic Mechanism of Dipeptidyl Peptidase IV

Human dipeptidyl peptidase IV (DPP-IV) is a ubiquitously expressed type II transmembrane serine protease. It cleaves the penultimate positioned prolyl bonds at the N terminus of physiologically important peptides such as the incretin hormones glucagon-like peptide 1 and glucose-dependent insulinotropic peptide. In this study, we have characterized different active site mutants. The Y547F mutant as well as the catalytic triad mutants S630A, D708A, and H740L showed less than 1% wild type activity. X-ray crystal structure analysis of the Y547F mutant revealed no overall changes compared with wild type apoDPP-IV, except the ablation of the hydroxyl group of Tyr(547) and a water molecule positioned in close proximity to Tyr(547). To elucidate further the reaction mechanism, we determined the crystal structure of DPP-IV in complex with diisopropyl fluorophosphate, mimicking the tetrahedral intermediate. The kinetic and structural findings of the tyrosine residue are discussed in relation to the catalytic mechanism of DPP-IV and to the inhibitory mechanism of the 2-cyanopyrrolidine class of potent DPP-IV inhibitors, proposing an explanation for the specificity of this class of inhibitors for the S9b family among serine proteases.

Structure of Kre2p/Mnt1p: A YEAST α1,2-MANNOSYLTRANSFERASE INVOLVED IN MANNOPROTEIN BIOSYNTHESIS

Kre2p/Mnt1p is a Golgi alpha1,2-mannosyltransferase involved in the biosynthesis of Saccharomyces cerevisiae cell wall glycoproteins. The protein belongs to glycosyltransferase family 15, a member of which has been implicated in virulence of Candida albicans. We present the 2.0 A crystal structures of the catalytic domain of Kre2p/Mnt1p and its binary and ternary complexes with GDP/Mn(2+) and GDP/Mn(2+)/acceptor methyl-alpha-mannoside. The protein has a mixed alpha/beta fold similar to the glycosyltransferase-A (GT-A) fold. Although the GDP-mannose donor was used in the crystallization experiments and the GDP moiety is bound tightly to the active site, the mannose is not visible in the electron density. The manganese is coordinated by a modified DXD motif (EPD), with only the first glutamate involved in a direct interaction. The position of the donor mannose was modeled using the binary and ternary complexes of other GT-A enzymes. The C1" of the modeled donor mannose is within hydrogen-bonding distance of both the hydroxyl of Tyr(220) and the O2 of the acceptor mannose. The O2 of the acceptor mannose is also within hydrogen bond distance of the hydroxyl of Tyr(220). The structures, modeling, site-directed mutagenesis, and kinetic analysis suggest two possible catalytic mechanisms. Either a double-displacement mechanism with the hydroxyl of Tyr(220) as the potential nucleophile or alternatively, an S(N)i-like mechanism with Tyr(220) positioning the substrates for catalysis. The importance of Tyr(220) in both mechanisms is highlighted by a 3000-fold reduction in k(cat) in the Y220F mutant.

GTP-Yeast Actin

Because of the apparently greater conformational flexibility of yeast versus muscle actin and the ability of other members in the actin protein superfamily to efficiently use both ATP and GTP, we assessed the ability of yeast actin to function with GTP. Etheno-ATP exchange studies showed that the binding of GTP to yeast actin is about 1/9 as tight as that of ATP in contrast to the 1/1,240 ratio for muscle actin. Proteolysis of GTP-bound G-yeast actin suggests that the conformation of subdomain 2 is very much like that of ATP-bound actin, but CD studies show that GTP-bound actin is less thermostable than ATP-bound actin. GTP-actin polymerizes with an apparent critical concentration of 1.5 microm, higher than that of ATP-actin (0.3 microm) although filament structures observed by electron microscopy were similar. Yeast actin hydrolyzes GTP in a polymerization-dependent manner, and GTP-bound F-actin decorates with the myosin S1. Conversion of Phe(306) in the nucleotide binding site to the Tyr found in muscle actin raised the nucleotide discrimination ratio from the 1/9 of wild-type actin to 1/125. This result agrees with modeling that predicts that removal of the Tyr hydroxyl will create a space for the C2 amino group of the GTP guanine.

Implication by site-directed mutagenesis of Arg 314 and Tyr 316 in the coenzyme site of pig mitochondrial NADP-dependent isocitrate dehydrogenase

Sequence alignment of pig mitochondrial NADP-dependent isocitrate dehydrogenase with eukaryotic (human, rat, and yeast) and Escherichia coli isocitrate dehydrogenases reveals that Tyr 316 is completely conserved and is equivalent to the E. coli Tyr 345, which interacts with the 2 ′-phosphate of NADP in the crystal structure [Hurley et al., Biochemistry 30 (1991) 8671–8678]. Lys 321 is also completely conserved in the five isocitrate dehydrogenases. Either an arginine or lysine residue is found among the enzymes from other species at the position corresponding to the pig enzyme Arg 314. While Arg 323 is not conserved among all species, its proximity to the coenzyme site makes it a good candidate for investigation. The importance of these four amino acids to the function of pig mitochondrial NADP-isocitrate dehydrogenase was studied by site-directed mutagenesis. Mutants (R314Q, Y316F, Y316L, K321Q, and R323Q) were generated by a megaprimer polymerase chain reaction method. Wild-type and mutant enzymes were expressed in E. coli and purified to homogeneity. All mutant and wild-type enzymes exhibited comparable molecular weights indicative of the dimeric enzyme. Mutations do not cause an appreciable change in enzyme secondary structure as revealed by circular dichroism measurements. The kinetic parameters ( V max and K M values) of K321Q and R323Q are similar to those of wild-type, indicating that Lys 321 and Arg 323 are not involved in enzyme function. R314Q exhibits a 10-fold increase in K M for NADP as compared to that of wild-type, while they have comparable V max values. These results suggest that Arg 314 contributes to the affinity between the enzyme and NADP. The hydroxyl group of Tyr 316 is not required for enzyme function since Y316F exhibits similar kinetic parameters to those of wild-type. Y316L shows a 4-fold increase in K M for NADP and a decrease in V max as compared to wild-type, suggesting that the aromatic ring of the Tyr of isocitrate dehydrogenase contributes to the affinity for coenzyme, as well as to catalysis. The K i for NAD of R314Q, Y316F, and Y316L is comparable to that of wild-type, indicating that the Arg 314 and Tyr 316 may be located near the 2 ′-phosphate of enzyme-bound NADP.

Arg362 and Tyr365 of the Botulinum Neurotoxin Type A Light Chain Are Involved in Transition State Stabilization

The botulinum neurotoxin type A (BoNT/A) light chain (LC) acts as zinc endopeptidase. The X-ray structure of the toxin demonstrated that Zn(2+) is coordinated by His(222) and His(226) of the Zn(2+) binding motif HisGluXXHis and Glu(261), whereas Glu(223) coordinates the water molecule required for hydrolysis as the fourth ligand. Recent analysis of a cocrystal of the BoNT/B LC and its substrate synaptobrevin 2 suggested that Arg(362) and Tyr(365) of the homologous BoNT/A may be directly involved in catalysis. Their role and that of Glu(350) which is also found in the vicinity to the active site were analyzed by site-directed mutagenesis. Various replacements of Arg(362) and substitution of Tyr(365) with Phe resulted in 79- and 34-fold lower k(cat)/K(m) values, respectively. These changes were provoked by decreased catalytic rates (k(cat)) and not by alterations of ground state substrate binding as evidenced by largely unchanged K(d) and K(m) values. None of these mutations affected the overall secondary structure or zinc content of the LC. These findings suggest that the guanidino group of Arg(362) and the hydroxyl group of Tyr(365) together accomplish transition state stabilization as was proposed for thermolysin, being the prototypical member of the gluzincin superfamily of metalloproteases. Mutation of Glu(350) dramatically diminished the hydrolytic activity which must partly be attributed to an altered active site fine structure as demonstrated by an increased sensitivity toward heat-induced denaturing and a lower Zn(2+) binding affinity. Glu(350) apparently occupies a central position in the active site and presumably positions His(222) and Arg(362).

Tyrosine 220 in the 5th Transmembrane Domain of the Neuromedin B Receptor Is Critical for the High Selectivity of the Peptoid Antagonist PD168368

Peptoid antagonists are increasingly being described for G protein-coupled receptors; however, little is known about the molecular basis of their binding. Recently, the peptoid PD168368 was found to be a potent selective neuromedin B receptor (NMBR) antagonist. To investigate the molecular basis for its selectivity for the NMBR over the closely related receptor for gastrin-releasing peptide (GRPR), we used a chimeric receptor approach and a site-directed mutagenesis approach. Mutated receptors were transiently expressed in Balb 3T3. The extracellular domains of the NMBR were not important for the selectivity of PD168368. However, substitution of the 5th upper transmembrane domain (uTM5) of the NMBR by the comparable GRPR domains decreased the affinity 16-fold. When the reverse study was performed by substituting the uTM5 of NMBR into the GRPR, a 9-fold increase in affinity occurred. Each of the 4 amino acids that differed between NMBR and GRPR in the uTM5 region were exchanged, but only the substitution of Phe(220) for Tyr in the NMBR caused a decrease in affinity. When the reverse study was performed to attempt to demonstrate a gain of affinity in the GRPR, the substitution of Tyr(219) for Phe caused an increase in affinity. These results suggest that the hydroxyl group of Tyr(220) in uTM5 of NMBR plays a critical role for high selectivity of PD168368 for NMBR over GRPR. Receptor and ligand modeling suggests that the hydroxyl of the Tyr(220) interacts with nitrophenyl group of PD168368 likely primarily by hydrogen bonding. This result shows the selectivity of the peptoid PD168368, similar to that reported for numerous non-peptide analogues with other G protein-coupled receptors, is primarily dependent on interaction with transmembrane amino acids.

Role of tyrosine 65 in the mechanism of serine hydroxymethyltransferase.

Crystal structures of human and rabbit cytosolic serine hydroxymethyltransferase have shown that Tyr65 is likely to be a key residue in the mechanism of the enzyme. In the ternary complex of Escherichia coli serine hydroxymethyltransferase with glycine and 5-formyltetrahydrofolate, the hydroxyl of Tyr65 is one of four enzyme side chains within hydrogen-bonding distance of the carboxylate group of the substrate glycine. To probe the role of Tyr65 it was changed by site-directed mutagenesis to Phe65. The three-dimensional structure of the Y65F site mutant was determined and shown to be isomorphous with the wild-type enzyme except for the missing Tyr hydroxyl group. The kinetic properties of this mutant enzyme in catalyzing reactions with serine, glycine, allothreonine, D- and L-alanine, and 5,10-methenyltetrahydrofolate substrates were determined. The properties of the enzyme with D- and L-alanine, glycine in the absence of tetrahydrofolate, and 5, 10-methenyltetrahydrofolate were not significantly changed. However, catalytic activity was greatly decreased for serine and allothreonine cleavage and for the solvent alpha-proton exchange of glycine in the presence of tetrahydrofolate. The decreased catalytic activity for these reactions could be explained by a greater than 2 orders of magnitude increase in affinity of Y65F mutant serine hydroxymethyltransferase for these amino acids bound as the external aldimine. These data are consistent with a role for the Tyr65 hydroxyl group in the conversion of a closed active site to an open structure.

Ab initio calculations on hidden modulators of theta class glutathione transferase activity.

The glutathione transferases decrease the pKa of glutathione, allowing its deprotonation and the formation of the more reactive thiolate anion. The thiolate is maintained in the active site through a weak conventional hydrogen bond first sphere interaction donated by a Tyr hydroxyl in the Alpha, Mu, Pi, and Sigma glutathione transferase classes that can be modified by other second sphere or indirect thiolate contacts. However, the Theta and Delta class isoforms use a Ser hydroxyl for stabilizing the GSH thiolate, and as such, have a different chemical system compared with that of the Tyr possessed by other classes. We have used high level ab initio methods to investigate this interaction by using a simple methanol methanethiol system as a model. The hydrogen bond strength of this initial first sphere interaction was calculated to be less than that of the Tyr interaction. A putative second sphere interaction exists in the Theta and Delta class structures between Cys or Ser-14 and Ser-11 in the mammalian Theta subclass 1 and 2, respectively. The effect of this interaction on the first sphere interaction has also been investigated and found to significantly increase the energy of the bond.

Binding of Alkylurea Inhibitors to Epoxide Hydrolase Implicates Active Site Tyrosines in Substrate Activation

The structures of two alkylurea inhibitors complexed with murine soluble epoxide hydrolase have been determined by x-ray crystallographic methods. The alkyl substituents of each inhibitor make extensive hydrophobic contacts in the soluble epoxide hydrolase active site, and each urea carbonyl oxygen accepts hydrogen bonds from the phenolic hydroxyl groups of Tyr(381) and Tyr(465). These hydrogen bond interactions suggest that Tyr(381) and/or Tyr(465) are general acid catalysts that facilitate epoxide ring opening in the first step of the hydrolysis reaction; Tyr(465) is highly conserved among all epoxide hydrolases, and Tyr(381) is conserved among the soluble epoxide hydrolases. In one enzyme-inhibitor complex, the urea carbonyl oxygen additionally interacts with Gln(382). If a comparable interaction occurs in catalysis, then Gln(382) may provide electrostatic stabilization of partial negative charge on the epoxide oxygen. The carboxylate side chain of Asp(333) accepts a hydrogen bond from one of the urea NH groups in each enzyme-inhibitor complex. Because Asp(333) is the catalytic nucleophile, its interaction with the partial positive charge on the urea NH group mimics its approach toward the partial positive charge on the electrophilic carbon of an epoxide substrate. Accordingly, alkylurea inhibitors mimic features encountered in the reaction coordinate of epoxide ring opening, and a structure-based mechanism is proposed for leukotoxin epoxide hydrolysis.

Binding pockets of the beta(1)- and beta(2)-adrenergic receptors for subtype-selective agonists.

We examined the subtype-selective binding site of the beta-adrenergic receptors (betaARs). The beta(1)/beta(2)-chimeric receptors showed the importance of the second and seventh transmembrane domains (TM2 and TM7) of the beta(2)AR for the binding of the beta(2)-selective agonists such as formoterol and procaterol. Alanine-substituted mutants of TM7 of the beta(2)AR showed that Tyr(308,) located at the top of TM7, mainly contributed to beta(2) selectivity. However, Tyr(308) interacted with formoterol and procaterol in two different ways. The results of Ala- and Phe-substituted mutants indicated that the phenyl group of Tyr(308) interacted with the phenyl group in the N-substituent of formoterol (hydrophobic interaction), and the hydroxyl group of Tyr(308) interacted with the protonated amine of procaterol (hydrophilic interaction). In contrast to beta(2)AR, TM2 is a major determinant that beta(1)-selective agonists such as denopamine and T-0509 bound the beta(1)AR with high affinity. Three amino acids (Leu(110), Thr(117), and Val(120)) in TM2 of the beta(1)AR were identified as major determinants for beta(1)-selective binding of these agonists. Three-dimensional models built on the basis of the predicted structure of rhodopsin showed that Tyr(308) of the beta(2)AR covered the binding pocket formed by TM2 and TM7 from the upper side, and Thr(117) of the beta(1)AR located in the middle of the binding pocket to provide a hydrogen bonding for the beta(1)-selective agonists. These data indicate that TM2 and TM7 of the betaAR formed the binding pocket that binds the betaAR subtype-selective agonists with high affinity.

Amino acids and peptides. LIV. Application of 2-adamantyl derivatives as protecting groups to the synthesis of peptide fragments related to Sulfolobus solifataricus ribonuclease. I.

The 2-adamantyloxycarbonyl group was employed for the protection of the epsilon-amino group of Lys and the hydroxyl group of Tyr, and the 2-adamantyl ester was employed for the protection of the beta-carboxyl group of Asp in order to construct eight peptide segments as building blocks for the preparation of peptide fragments related to the sequence of Sulfolobus solifataricus Ribonuclease. The usefulness of the above protecting groups developed in our laboratory was confirmed.

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.

Solution structure of glutathione bound to human glutathione transferase P1-1: comparison of NMR measurements with the crystal structure.

The conformation of the bound glutathione (GSH) in the active site of the human glutathione transferase P1-1 (EC 2.5.1.18) has been studied by transferred NOE measurements and compared with those obtained by X-ray diffraction data. Two-dimensional TRNOESY and TRROESY experiments have been performed under fast-exchange conditions. The family of GSH conformers, compatible with TRNOE distance constraints, shows a backbone structure very similar to the crystal model. Interesting differences have been found in the side chain regions. After restrained energy minimization of a representative NMR conformer in the active site, the sulfur atom is not found in hydrogen-bonding distance of the hydroxyl group of Tyr 7. This situation is similar to the one observed in an "atypical" crystal complex grown at low pH and low temperature. The NMR conformers display also a poorly defined structure of the glutamyl moiety, and the presence of an unexpected intermolecular NOE could indicate a different interaction of this substrate portion with the G-site. The NMR data seem to provide a snapshot of GSH in a precomplex where the GSH glutamyl end is bound in a different fashion. The existence of this precomplex is supported by pre-steady-state kinetic experiments [Caccuri, A. M., Lo Bello, M., Nuccetelli, M., Nicotra, M., Rossi, P., Antonini, G., Federici, G., and Ricci, G. (1998) Biochemistry 37, 3028-3034] and preliminary time-resolved fluorescence data.

The refined crystal structure of the 3C gene product from hepatitis A virus: specific proteinase activity and RNA recognition.

The virally encoded 3C proteinases of picornaviruses process the polyprotein produced by the translation of polycistronic viral mRNA. The X-ray crystallographic structure of a catalytically active mutant of the hepatitis A virus (HAV) 3C proteinase (C24S) has been determined. Crystals of this mutant of HAV 3C are triclinic with unit cell dimensions a = 53.6 A, b = 53.5 A, c = 53.2 A, alpha = 99.1 degrees, beta = 129.0 degrees, and gamma = 103.3 degrees. There are two molecules of HAV 3C in the unit cell of this crystal form. The structure has been refined to an R factor of 0.211 (Rfree = 0.265) at 2.0-A resolution. Both molecules fold into the characteristic two-domain structure of the chymotrypsin-like serine proteinases. The active-site and substrate-binding regions are located in a surface groove between the two beta-barrel domains. The catalytic Cys 172 S(gamma) and His 44 N(epsilon2) are separated by 3.9 A; the oxyanion hole adopts the same conformation as that seen in the serine proteinases. The side chain of Asp 84, the residue expected to form the third member of the catalytic triad, is pointed away from the side chain of His 44 and is locked in an ion pair interaction with the epsilon-amino group of Lys 202. A water molecule is hydrogen bonded to His 44 N(delta1). The side-chain phenolic hydroxyl group of Tyr 143 is close to this water and to His 44 N(delta1) and may be negatively charged. The glutamine specificity for P1 residues of substrate cleavage sites is attributed to the presence of a highly conserved His 191 in the S1 pocket. A very unusual environment of two water molecules and a buried glutamate contribute to the imidazole tautomer believed to be important in the P1 specificity. HAV 3C proteinase has the conserved RNA recognition sequence KFRDI located in the interdomain connection loop on the side of the molecule diametrically opposite the proteolytic site. This segment of polypeptide is located between the N- and C-terminal helices, and its conformation results in the formation of a well-defined surface with a strongly charged electrostatic potential. Presumably, this surface of HAV 3C participates in the recognition of the 5' and 3' nontranslated regions of the RNA genome during viral replication.