Spectroscopic characterization of soybean lipoxygenase-1 mutants: the role of second coordination sphere residues in the regulation of enzyme activity

Lipoxygenases are non-heme iron enzymes, which catalyze the stereo- and regiospecific hydroperoxidation of unsaturated fatty acids. Spectroscopic studies on soybean lipoxygenase have shown that the ferrous form of the enzyme is a mixture of five- and six-coordinate species (40 and 60%, respectively). Addition of substrate leads to a purely six-coordinate form. A series of mutations in the second coordination sphere (Q697E, Q697N, Q495A, and Q495E) were generated, and the structures of the mutants were solved by crystallography [Tomchick et al. (2001) Biochemistry 40, 7509-7517]. While this study clearly showed the contribution of H-bond interactions between the first and the second coordination spheres in catalysis, no correlation with the coordination environment of the Fe(II) was observed. A recent study using density-functional theory [Lehnert and Solomon (2002) J. Biol. Inorg. Chem. 8, 294-305] indicated that coordination flexibility, involving the Asn694 ligand, is regulated via H-bond interactions. In this paper, we investigate the solution structures of the second coordination sphere mutants using CD and MCD spectroscopy since these techniques are more sensitive indicators of the first coordination sphere ligation of Fe(II) systems. Our data demonstrate that the iron coordination environment directly relates to activity, with the mutations that have the ability to form a five-coordinate/six-coordinate mixture being more active. We propose that the H-bond between the weak Asn694 ligand and the Gln697 plays a key role in the modulation of the coordination flexibility of Asn694, and thus, is crucial for the regulation of enzyme reactivity.     

Stabilization of the transition state for the transfer of tyrosine to tRNA(Tyr) by tyrosyl-tRNA synthetase

Aminoacylation of tRNA(Tyr) involves two steps: (1) tyrosine activation to form the tyrosyl-adenylate intermediate; and (2) transfer of tyrosine from the tyrosyl-adenylate intermediate to tRNA(Tyr). In Bacillus stearothermophilus tyrosyl-tRNA synthetase, Asp78, Tyr169, and Gln173 have been shown to form hydrogen bonds with the alpha-ammonium group of the tyrosine substrate during the first step of the aminoacylation reaction. Asp194 and Gln195 stabilize the transition state complex for the first step of the reaction by hydrogen bonding with the 2'-hydroxyl group of AMP and the carboxylate oxygen atom of tyrosine, respectively. Here, the roles that Asp78, Tyr169, Gln173, Asp194, and Gln195 play in catalysis of the second step of the reaction are investigated. Pre-steady-state kinetic analyses of alanine variants at each of these positions shows that while the replacement of Gln173 by alanine does not affect the initial binding of the tRNA(Tyr) substrate, it destabilizes the transition state complex for the second step of the reaction by 2.3 kcal/mol. None of the other alanine substitutions affects either the initial binding of the tRNA(Tyr) substrate or the stability of the transition state for the second step of the aminoacylation reaction. Taken together, the results presented here and the accompanying paper are consistent with a concerted reaction mechanism for the transfer of tyrosine to tRNA(Tyr), and suggest that catalysis of the second step of tRNA(Tyr) aminoacylation involves stabilization of a transition state in which the scissile acylphosphate bond of the tyrosyl-adenylate species is strained. Cleavage of the scissile bond on the breakdown of the transition state alleviates this strain.     

Mutants that alter the covalent structure of catalase hydroperoxidase II from Escherichia coli.

The three-dimensional structures of two HPII variants, V169C and H392Q, have been determined at resolutions of 1.8 and 2.1 A, respectively. The V169C variant contains a new type of covalent bond between the sulfur atom of Cys(169) and a carbon atom on the imidazole ring of the essential His(128). This variant enzyme has only residual catalytic activity and contains heme b. The chain of water molecules visible in the main channel may reflect the organization of the hydrogen peroxide substrates in the active enzyme. Two alternative mechanisms, involving either compound I or free radical intermediates, are presented to explain the formation of the Cys-His covalent bond. The H392Q and H392E variants exhibit 75 and 25% of native catalytic activity, respectively. The Gln(392) variant contains only heme b, whereas the Glu(392) variant contains a mixture of heme b and cis and trans isomers of heme d, suggesting of a role for this residue in heme conversion. Replacement of either Gln(419) and Ser(414), both of which interact with the heme, affected the cis:trans ratio of spirolactone heme d. Implications for the heme oxidation mechanism and the His-Tyr bond formation in HPII are considered.     

Residue 259 in protein-tyrosine phosphatase PTP1B and PTPalpha determines the flexibility of glutamine 262

To study the flexibility of the substrate-binding site and in particular of Gln262, we have performed adiabatic conformational search and molecular dynamics simulations on the crystal structure of the catalytic domain of wild-type protein-tyrosine phosphatase (PTP) 1B, a mutant PTP1B(R47V,D48N,M258C,G259Q), and a model of the catalytically active form of PTPalpha. For each molecule two cases were modeled: the Michaelis-Menten complex with the substrate analogue p-nitrophenyl phosphate (p-PNPP) bound to the active site and the cysteine-phosphor complex, each corresponding to the first and second step of the phosphate hydrolysis. Analyses of the trajectories revealed that in the cysteine-phosphor complex of PTP1B, Gln262 oscillates freely between the bound phosphate group and Gly259 frequently forming, as observed in the crystal structure, a hydrogen bond with the backbone oxygen of Gly259. In contrast, the movement of Gln262 is restricted in PTPalpha and the mutant due to interactions with Gln259 reducing the frequency of the oscillation of Gln262 and thereby delaying the positioning of this residue for the second step in the catalysis, as reflected experimentally by a reduction in k(cat). Additionally, in the simulation with the Michaelis-Menten complexes, we found that a glutamine in position 259 induces steric hindrance by pushing the Gln262 side chain further toward the substrate and thereby negatively affecting K(m) as indicated by kinetic studies. Detailed analysis of the water structure around Gln262 and the active site Cys215 reveals that the probability of finding a water molecule correctly positioned for catalysis is much larger in PTP1B than in PTP1B(R47V,D48N,M258C,G259Q) and PTPalpha, in accordance with experiments.     

Residues glutamate 216 and aspartate 301 are key determinants of substrate specificity and product regioselectivity in cytochrome P450 2D6

Cytochrome P450 2D6 (CYP2D6) metabolizes a wide range of therapeutic drugs. CYP2D6 substrates typically contain a basic nitrogen atom, and the active-site residue Asp-301 has been implicated in substrate recognition through electrostatic interactions. Our recent computational models point to a predominantly structural role for Asp-301 in loop positioning (Kirton, S. B., Kemp, C. A., Tomkinson, N. P., St.-Gallay, S., and Sutcliffe, M. J. (2002) Proteins 49, 216-231) and suggest a second acidic residue, Glu-216, as a key determinant in the binding of basic substrates. We have evaluated the role of Glu-216 in substrate recognition, along with Asp-301, by site-directed mutagenesis. Reversal of the Glu-216 charge to Lys or substitution with neutral residues (Gln, Phe, or Leu) greatly decreased the affinity (K(m) values increased 10-100-fold) for the classical basic nitrogen-containing substrates bufuralol and dextromethorphan. Altered binding was also manifested in significant differences in regiospecificity with respect to dextromethorphan, producing enzymes with no preference for N-demethylation versus O-demethylation (E216K and E216F). Neutralization of Asp-301 to Gln and Asn had similarly profound effects on substrate binding and regioselectivity. Intriguingly, removal of the negative charge from either 216 or 301 produced enzymes (E216A, E216K, and D301Q) with elevated levels (50-75-fold) of catalytic activity toward diclofenac, a carboxylate-containing CYP2C9 substrate that lacks a basic nitrogen atom. Activity was increased still further (>1000-fold) upon neutralization of both residues (E216Q/D301Q). The kinetic parameters for diclofenac (K(m) 108 microm, k(cat) 5 min(-1)) along with nifedipine (K(m) 28 microm, k(cat) 2 min(-1)) and tolbutamide (K(m) 315 microm, k(cat) 1 min(-1)), which are not normally substrates for CYP2D6, were within an order of magnitude of those observed with CYP3A4 or CYP2C9. Neutralizing both Glu-216 and Asp-301 thus effectively alters substrate recognition illustrating the central role of the negative charges provided by both residues in defining the specificity of CYP2D6 toward substrates containing a basic nitrogen.     

Structure of 3,4-dihydroxy-2-butanone 4-phosphate synthase from Methanococcus jannaschii in complex with divalent metal ions and the substrate ribulose 5-phosphate: implications for the catalytic mechanism

Skeletal rearrangements of carbohydrates are crucial for many biosynthetic pathways. In riboflavin biosynthesis ribulose 5-phosphate is converted into 3,4-dihydroxy-2-butanone 4-phosphate while its C4 atom is released as formate in a sequence of metal-dependent reactions. Here, we present the crystal structure of Methanococcus jannaschii 3,4-dihydroxy-2-butanone 4-phosphate synthase in complex with the substrate ribulose 5-phosphate at a dimetal center presumably consisting of non-catalytic zinc and calcium ions at 1.7-A resolution. The carbonyl group (O2) and two out of three free hydroxyl groups (OH3 and OH4) of the substrate are metal-coordinated. We correlate previous mutational studies on this enzyme with the present structural results. Residues of the first coordination sphere involved in metal binding are indispensable for catalytic activity. Only Glu-185 of the second coordination sphere cannot be replaced without complete loss of activity. It contacts the C3 hydrogen atom directly and probably initiates enediol formation in concert with both metal ions to start the reaction sequence. Mechanistic similarities to Rubisco acting on the similar substrate ribulose 1,5-diphosphate in carbon dioxide fixation as well as other carbohydrate (reducto-) isomerases are discussed.     

Contribution of Gln9 and Phe80 to substrate binding in ribonuclease MC1 from bitter gourd seeds.

Ribonuclease MC1 (RNase MC1) isolated from bitter gourd (Momordica charantia) seeds specifically cleaves phosphodiester bonds on the 5'-side of uridine. The crystal structures of RNase MC1 in complex with 2'-UMP or 3'-UMP reveal that Gln9, Asn71, Leu73, and Phe80 are involved in uridine binding by hydrogen bonding and hydrophobic interactions [Suzuki et al. (2000) Biochem. Biophys. Res. Commun. 275, 572-576]. To evaluate the contribution of Gln9 and Phe80 to uridine binding, Gln9 was replaced with Ala, Phe, Glu, or His, and Phe80 with Ala by site-directed mutagenesis. The kinetic properties of the resulting mutant enzymes were characterized using cytidylyl-3',5'-uridine (CpU) as a substrate. The mutant Q9A exhibited a 3.7-fold increased K(m) and 27.6-fold decreased k(cat), while three other mutations, Q9F, Q9E, and Q9H, predominantly affected the k(cat) value. Replacing Phe80 with Ala drastically reduced the catalytic efficiency (k(cat)/K(m)) with a minimum K(m) value equal to 8 mM. It was further found that the hydrolytic activities of the mutants toward cytidine-2',3'-cyclic monophosphate (cCMP) were reduced. These results demonstrate that Gln9 and Phe80 play essential roles not only in uridine binding but also in hydrolytic activity. Moreover, we produced double Ala substituted mutants at Gln9, Asn71, Leu73, and Phe80, and compared their kinetic properties with those of the corresponding single mutants. The results suggest that these four residues may contribute to uridine binding in a mutually independent manner.     

Glutamine-181 is crucial in the enzymatic activity and substrate specificity of human endoplasmic-reticulum aminopeptidase-1

ERAP-1 (endoplasmic-reticulum aminopeptidase-1) is a multifunctional enzyme with roles in the regulation of blood pressure, angiogenesis and the presentation of antigens to MHC class I molecules. Whereas the enzyme shows restricted specificity toward synthetic substrates, its substrate specificity toward natural peptides is rather broad. Because of the pathophysiological significance of ERAP-1, it is important to elucidate the molecular basis of its enzymatic action. In the present study we used site-directed mutagenesis to identify residues affecting the substrate specificity of human ERAP-1 and identified Gln(181) as important for enzymatic activity and substrate specificity. Replacement of Gln(181) by aspartic acid resulted in a significant change in substrate specificity, with Q181D ERAP-1 showing a preference for basic amino acids. In addition, Q181D ERAP-1 cleaved natural peptides possessing a basic amino acid at the N-terminal end more efficiently than did the wild-type enzyme, whereas its cleavage of peptides with a non-basic amino acid was significantly reduced. Another mutant enzyme, Q181E, also revealed some preference for peptides with a basic N-terminal amino acid, although it had little hydrolytic activity toward the synthetic peptides tested. Other mutant enzymes, including Q181N and Q181A ERAP-1s, revealed little enzymatic activity toward synthetic or peptide substrates. These results indicate that Gln(181) is critical for the enzymatic activity and substrate specificity of ERAP-1.     

Catalytic residues and substrate specificity of scytalidoglutamic peptidase, the first member of the eqolisin in family (G1) of peptidases

Scytalidoglutamic peptidase (SGP) is the first-discovered member of the eqolisin family of peptidases with a unique structure and a presumed novel catalytic dyad (E136 and Q53) [Fujinaga et al., PNAS 101 (2004) 3364-3369]. Mutants of SGP, E136A, Q53A, and Q53E lost both the autoprocessing and enzymatic activities of the wild-type enzyme. Coupled with the results from the structural analysis of SGP, Glu136 and Gln53 were identified as the catalytic residues. The substrate specificity of SGP is unique, particularly, in the preference at the P3 (basic amino acid), P1' (small a.a.), and P3' (basic a.a.) positions. Superior substrates and inhibitors have been synthesized for kinetic studies based on the results reported here. kcat, Km, and kcat/Km of SGP for D-Dap(MeNHBz)-GFKFF*ALRK(Dnp)-D-R-D-R were 34.8 s-1, 0.065 microM, and 535 microM-1 s-1, respectively. Ki of Ac-FKF-(3S,4S)-phenylstatinyl-LR-NH2 for SGP was 1.2x10(-10) M. Taken together, we can conclude that SGP has not only structural and catalytic novelties but also a unique subsite structure.     

Structural and mutagenesis studies of leishmania triosephosphate isomerase: a point mutation can convert a mesophilic enzyme into a superstable enzyme without losing catalytic power

The dimeric enzyme triosephosphate isomerase (TIM) has a very tight and rigid dimer interface. At this interface a critical hydrogen bond is formed between the main chain oxygen atom of the catalytic residue Lys13 and the completely buried side chain of Gln65 (of the same subunit). The sequence of Leishmania mexicana TIM, closely related to Trypanosoma brucei TIM (68% sequence identity), shows that this highly conserved glutamine has been replaced by a glutamate. Therefore, the 1.8 A crystal structure of leishmania TIM (at pH 5.9) was determined. The comparison with the structure of trypanosomal TIM shows no rearrangements in the vicinity of Glu65, suggesting that its side chain is protonated and is hydrogen bonded to the main chain oxygen of Lys13. Ionization of this glutamic acid side chain causes a pH-dependent decrease in the thermal stability of leishmania TIM. The presence of this glutamate, also in its protonated state, disrupts to some extent the conserved hydrogen bond network, as seen in all other TIMs. Restoration of the hydrogen bonding network by its mutation to glutamine in the E65Q variant of leishmania TIM results in much higher stability; for example, at pH 7, the apparent melting temperature increases by 26 degrees C (57 degrees C for leishmania TIM to 83 degrees C for the E65Q variant). This mutation does not affect the kinetic properties, showing that even point mutations can convert a mesophilic enzyme into a superstable enzyme without losing catalytic power at the mesophilic temperature.     

Deuterium isotope effects during carbon-hydrogen bond cleavage by trimethylamine dehydrogenase. Implications for mechanism and vibrationally assisted hydrogen tunneling in wild-type and mutant enzymes

His-172 and Tyr-169 are components of a triad in the active site of trimethylamine dehydrogenase (TMADH) comprising Asp-267, His-172, and Tyr-169. Stopped-flow kinetic studies with trimethylamine as substrate have indicated that mutation of His-172 to Gln reduces the limiting rate constant for flavin reduction approximately 10-fold (Basran, J., Sutcliffe, M. J., Hille, R., and Scrutton, N. S. (1999) Biochem. J. 341, 307-314). A kinetic isotope effect (KIE = k(H)/k(D)) accompanies flavin reduction by H172Q TMADH, the magnitude of which varies significantly with solution pH. With trimethylamine, flavin reduction by H172Q TMADH is controlled by a single macroscopic ionization (pK(a) = 6.8 +/- 0.1). This ionization is perturbed (pK(a) = 7.4 +/- 0.1) in reactions with perdeuterated trimethylamine and is responsible for the apparent variation in the KIE with solution pH. At pH 9.5, where the functional group controlling flavin reduction is fully ionized, the KIE is independent of temperature in the range 277-297 K, consistent with vibrationally assisted hydrogen tunneling during breakage of the substrate C-H bond. Y169F TMADH is approximately 4-fold more compromised than H172Q TMADH for hydrogen transfer, which occurs non-classically. Studies with Y169F TMADH suggest partial thermal excitation of substrate prior to hydrogen tunneling by a vibrationally assisted mechanism. Our studies illustrate the varied effects of compromising mutations on tunneling regimes in enzyme molecules.     

Structural asymmetry of phosphodiesterase-9, potential protonation of a glutamic acid, and role of the invariant glutamine

PDE9 inhibitors show potential for treatment of diseases such as diabetes. To help with discovery of PDE9 inhibitors, we performed mutagenesis, kinetic, crystallographic, and molecular dynamics analyses on the active site residues of Gln453 and its stabilizing partner Glu406. The crystal structures of the PDE9 Q453E mutant (PDE9Q453E) in complex with inhibitors IBMX and (S)-BAY73-6691 showed asymmetric binding of the inhibitors in two subunits of the PDE9Q453E dimer and also the significant positional change of the M-loop at the active site. The kinetic analysis of the Q453E and E406A mutants suggested that the invariant glutamine is critical for binding of substrates and inhibitors, but is unlikely to play a key role in the differentiation between substrates of cGMP and cAMP. The molecular dynamics simulations suggest that residue Glu406 may be protonated and may thus explain the hydrogen bond distance between two side chain oxygens of Glu453 and Glu406 in the crystal structure of the PDE9Q453E mutant. The information from these studies may be useful for design of PDE9 inhibitors.     

In silico study of structural determinants modulating the redox potential of Rigidoporus lignosus and other fungal laccases

Laccases are multicopper oxidases in which substrate oxidation takes place at the type-1 (T1) copper site. The redox potential (E (0)) significantly varies amongst members of the family and is a key parameter for substrate specificity. Despite sharing highly conserved features at the T1 copper site, laccases span a large range of E (0), suggesting that the influence of the metal secondary coordination sphere is important. In silico analysis of structural determinants modulating the E (0) of Rigidoporus lignosus and other fungal laccases indicated that different factors can be considered. First, the length of the T1 copper coordinating histidine bond is observed to be longer in high E (0) laccases than in low E (0) ones. The hydrophobic environment around the T1 copper site appeared as another important structural determinant in modulating the E (0), with a stronger hydrophobic environment correlating with higher E (0). The analysis of hydrogen bonding network (HBN) around the T1-binding pocket revealed that the amino acids building up the metal binding site strongly interact with neighbouring residues and contribute to the stabilization of the protein folds. Changes in these HBNs that modified the Cu1 preferred coordination geometry lead to an increase of E (0). The presence of axial ligands modulates the E (0) of T1 to different extent. Stacking interactions between aromatic residues located in the second coordination shell and the metal ion coordination histidine imidazole ring have also been identified as a factor that modulates the E (0). The electrostatic interactions between the T1 copper site and backbone carbonyl oxygen indicate that Cu1-CO=NH distance is longer in the high E (0) laccases. In short, the in silico study reported herein identifies several structural factors that may influence the E (0) of the examined laccases. Some of these are dependent on the nature of the coordination ligands at the T1 site, but others can be ascribed to the hydrophobic effects, HBNs, axial ligations, stacking and electrostatic interactions, not necessary directly interacting with the copper metal.     

Crystal structure of the ternary complex of the catalytic domain of human phenylalanine hydroxylase with tetrahydrobiopterin and 3-(2-thienyl)-L-alanine,and its implications for the mechanism of catalysis and substrate activation

Phenylalanine hydroxylase catalyzes the stereospecific hydroxylation of L-phenylalanine, the committed step in the degradation of this amino acid. We have solved the crystal structure of the ternary complex (hPheOH-Fe(II).BH(4).THA) of the catalytically active Fe(II) form of a truncated form (DeltaN1-102/DeltaC428-452) of human phenylalanine hydroxylase (hPheOH), using the catalytically active reduced cofactor 6(R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH(4)) and 3-(2-thienyl)-L-alanine (THA) as a substrate analogue. The analogue is bound in the second coordination sphere of the catalytic iron atom with the thiophene ring stacking against the imidazole group of His285 (average interplanar distance 3.8A) and with a network of hydrogen bonds and hydrophobic contacts. Binding of the analogue to the binary complex hPheOH-Fe(II).BH(4) triggers structural changes throughout the entire molecule, which adopts a slightly more compact structure. The largest change occurs in the loop region comprising residues 131-155, where the maximum r.m.s. displacement (9.6A) is at Tyr138. This loop is refolded, bringing the hydroxyl oxygen atom of Tyr138 18.5A closer to the iron atom and into the active site. The iron geometry is highly distorted square pyramidal, and Glu330 adopts a conformation different from that observed in the hPheOH-Fe(II).BH(4) structure, with bidentate iron coordination. BH(4) binds in the second coordination sphere of the catalytic iron atom, and is displaced 2.6A in the direction of Glu286 and the iron atom, relative to the hPheOH-Fe(II).BH(4) structure, thus changing its hydrogen bonding network. The active-site structure of the ternary complex gives new insight into the substrate specificity of the enzyme, notably the low affinity for L-tyrosine. Furthermore, the structure has implications both for the catalytic mechanism and the molecular basis for the activation of the full-length tetrameric enzyme by its substrate. The large conformational change, moving Tyr138 from a surface position into the active site, may reflect a possible functional role for this residue.     

Dissection of the structural and functional role of a conserved hydration site in RNase T1

The reoccurrence of water molecules in crystal structures of RNase T1 was investigated. Five waters were found to be invariant in RNase T1 as well as in six other related fungal RNases. The structural, dynamical, and functional characteristics of one of these conserved hydration sites (WAT1) were analyzed by protein engineering, X-ray crystallography, and (17)O and 2H nuclear magnetic relaxation dispersion (NMRD). The position of WAT1 and its surrounding hydrogen bond network are unaffected by deletions of two neighboring side chains. In the mutant Thr93Gln, the Gln93N epsilon2 nitrogen replaces WAT1 and participates in a similar hydrogen bond network involving Cys6, Asn9, Asp76, and Thr91. The ability of WAT1 to form four hydrogen bonds may explain why evolution has preserved a water molecule, rather than a side-chain atom, at the center of this intricate hydrogen bond network. Comparison of the (17)O NMRD profiles from wild-type and Thr93Gln RNase T1 yield a mean residence time of 7 ns at 27 degrees C and an orientational order parameter of 0.45. The effects of mutations around WAT1 on the kinetic parameters of RNase T1 are small but significant and probably relate to the dynamics of the active site.     

Substrate recognition mechanism of a glycosyltrehalose trehalohydrolase from Sulfolobus solfataricus KM1

Glycosyltrehalose trehalohydrolase (GTHase) is an α-amylase that cleaves the α-1,4 bond adjacent to the α-1,1 bond of maltooligosyltrehalose to release trehalose. To investigate the catalytic and substrate recognition mechanisms of GTHase, two residues, Asp252 (nucleophile) and Glu283 (general acid/base), located at the catalytic site of GTHase were mutated (Asp252→Ser (D252S), Glu (D252E) and Glu283→Gln (E283Q)), and the activity and structure of the enzyme were investigated. The E283Q, D252E, and D252S mutants showed only 0.04, 0.03, and 0.6% of enzymatic activity against the wild-type, respectively. The crystal structure of the E283Q mutant GTHase in complex with the substrate, maltotriosyltrehalose (G3-Tre), was determined to 2.6-Å resolution. The structure with G3-Tre indicated that GTHase has at least five substrate binding subsites and that Glu283 is the catalytic acid, and Asp252 is the nucleophile that attacks the C1 carbon in the glycosidic linkage of G3-Tre. The complex structure also revealed a scheme for substrate recognition by GTHase. Substrate recognition involves two unique interactions: stacking of Tyr325 with the terminal glucose ring of the trehalose moiety and perpendicularly placement of Trp215 to the pyranose rings at the subsites −1 and +1 glucose.     

Broad-line nuclear magnetic resonance studies of chloroperoxidase.

Chloroperoxidase, a heme glycoprotein isolated from the mold Caldariomyces fumago, was studied by NMR relaxation techniques. Interaction of the chloride ion substrate with the enzyme may be analyzed as consisting of at least three contributions: a weak interaction with the iron atom, nonspecific anion-protein interactions, and a specific interaction generated at low pH. The data indicate that a specific interaction, which develops in parallel with enzyme activity at low pH, does not occur at the iron atom first coordination sphere site. The results are summarized in terms of an enzymatic mechanism not involving chloride ion coordination to the iron atom.     

The critical role of a hydrogen bond between Gln63 and Trp104 in the blue-light sensing BLUF domain that controls AppA activity

AppA is a novel blue-light receptor that controls photosynthetic gene expression in the purple bacterium Rhodobacter sphaeroides. The photocycle reaction of the light-sensing domain, BLUF, is unique in the sense that a few hydrogen bond rearrangements are accompanied by only slight structural changes of the bound chromophore. However, the exact features of the hydrogen bond network around the active site are still the subject of some controversy. Here we present biochemical and genetic evidence showing that either Gln63 or Trp104 in the active site of the BLUF domain is crucial for light sensing, which in turn controls the antirepressor activity of AppA. Specifically, the Q63L and W104A mutants of AppA are insensitive to blue light in vivo and in vitro, and their activity is similar to that of the light-adapted wild-type AppA. Based on spectroscopic and structural information described previously, we conclude that light-dependent formation and breakage of the hydrogen bond between Gln63 and Trp104 are critical for the light-sensing mechanism of AppA.     

E230Q mutation of the catalytic subunit of cAMP-dependent protein kinase affects local structure and the binding of peptide inhibitor

The active site of the mammalian cAMP-dependent protein kinase catalytic subunit (C-subunit) has a cluster of nonconserved acidic residues-Glu127, Glu170, Glu203, Glu230, and Asp241-that are crucial for substrate recognition and binding. Studies have shown that the Glu230 to Gln mutant (E230Q) of the enzyme has physical properties similar to the wild-type enzyme and has decreased affinity for a short peptide substrate, Kemptide. However, recent experiments intended to crystallize ternary complex of the E230Q mutant with MgATP and protein kinase inhibitor (PKI) could only obtain crystals of the apo-enzyme of E230Q mutant. To deduce the possible mechanism that prevented ternary complex formation, we used the relaxed-complex method (Lin, J.-H., et al. J Am Chem Soc 2002, 24, 5632-5633) to study PKI binding to the E230Q mutant C-subunit. In the E230Q mutant, we observed local structural changes of the peptide binding site that correlated closely to the reduced PKI affinity. The structural changes occurred in the F-to-G helix loop and appeared to hinder PKI binding. Reduced electrostatic potential repulsion among Asp241 from the helix loop section and the other acidic residues in the peptide binding site appear to be responsible for the structural change.     

Symmetrical stabilization of bound Ca2+ ions in a cooperative pair of EF-hands through hydrogen bonding of coordinating water molecules in calbindin D(9k)

Water molecules are found to complete the Ca2+ coordination sphere when a protein fails to provide enough ligating oxygens. Hydrogen bonding of these water molecules to the protein backbone or side chains may contribute favorably to the Ca2+ affinity, as suggested in an earlier study of two calbindin D(9k) mutants [E60D and E60Q; Linse et al. (1994) Biochemistry 33, 12478-12486]. To investigate the generality of this conclusion, another side chain, Gln 22, which hydrogen bonds to a Ca2+-coordinating water molecule in calbindin D(9k), was mutated. Two calbindin D(9k) mutants, (Q22E+P43M) and (Q22N+P43M), were constructed to examine the interaction between Gln 22 and the water molecule in the C-terminal calcium binding site II. Shortening of the side chain, as in (Q22N+P43M), reduces the affinity of binding two calcium ions by a factor of 18 at low ionic strength, whereas introduction of a negative charge, as in (Q22E+P43M), leads to a 12-fold reduction. In 0.15 M KCl, a 7-fold reduction in affinity was observed for both mutants. The cooperativity of Ca2+ binding increases for (Q22E+P43M), while it decreases for (Q22N+P43M). The rates of Ca2+ dissociation are 5.5-fold higher for the double mutants than for P43M at low ionic strength. For both mutants, reduced strength of hydrogen bonding to calcium-coordinating water molecules is a likely explanation for the observed effects on Ca2+ affinity and dissociation. In the apo forms, the (Q22E+P43M) mutant has lower stability toward urea denaturation than (Q22N+P43M) and P43M. 2D (1)H NMR and crystallographic experiments suggest that the structure of (Q22E+P43M) and (Q22N+P43M) is unchanged relative to P43M, except for local perturbations in the loop regions.