Identification of a novel bond between a histidine and the essential tyrosine in catalase HPII of Escherichia coli.

A bond between the N delta of the imidazole ring of His 392 and the C beta of the essential Tyr 415 has been found in the refined crystal structure at 1.9 A resolution of catalase HPII of Escherichia coli. This novel type of covalent linkage is clearly defined in the electron density map of HPII and is confirmed by matrix-assisted laser desorption/ionization mass spectrometry analysis of tryptic digest mixtures. The geometry of the bond is compatible with both the sp3 hybridization of the C beta atom and the planarity of the imidazole ring. Two mutated variants of HPII active site residues, H128N and N201H, do not contain the His 392-Tyr 415 bond, and their crystal structures show that the imidazole ring of His 392 was rotated, in both cases, by 80 degrees relative to its position in HPII. These mutant forms of HPII are catalytically inactive and do not convert heme b to heme d, suggesting a relationship between the self-catalyzed heme conversion reaction and the formation of the His-Tyr linkage. A model coupling the two processes and involving the reaction of one molecule of H2O2 on the proximal side of the heme with compound 1 is proposed.     

Structure of catalase HPII from Escherichia coli at 1.9 A resolution

Catalase HPII from Escherichia coli, a homotetramer of subunits with 753 residues, is the largest known catalase. The structure of native HPII has been refined at 1.9 A resolution using X-ray synchrotron data collected from crystals flash-cooled with liquid nitrogen. The crystallographic agreement factors R and R(free) are respectively 16.6% and 21.0%. The asymmetric unit of the crystal contains a whole molecule that shows accurate 222-point group symmetry. The structure of the central part of the HPII subunit gives a root mean square deviation of 1.5 A for 477 equivalencies with beef liver catalase. Most of the additional 276 residues of HPII are located in either an extended N-terminal arm or in a C-terminal domain organized with a flavodoxin-like topology. A small number of mostly hydrophilic interactions stabilize the relative orientation between the C-terminal domain and the core of the enzyme. The heme component of HPII is a cis-hydroxychlorin gamma-spirolactone in an orientation that is flipped 180 degrees with respect to the orientation of the heme found in beef liver catalase. The proximal ligand of the heme is Tyr415 which is joined by a covalent bond between its Cbeta atom and the Ndelta atom of His392. Over 2,700 well-defined solvent molecules have been identified filling a complex network of cavities and channels formed inside the molecule. Two channels lead close to the distal side heme pocket of each subunit suggesting separate inlet and exhaust functions. The longest channel, that begins in an adjacent subunit, is over 50 A in length, and the second channel is about 30 A in length. A third channel reaching the heme proximal side may provide access for the substrate needed to catalyze the heme modification and His-Tyr bond formation. HPII does not bind NADPH and the equivalent region to the NADPH binding pocket of bovine catalase, partially occluded in HPII by residues 585-590, corresponds to the entrance to the second channel. The heme distal pocket contains two solvent molecules, and the one closer to the iron atom appears to exhibit high mobility or low occupancy compatible with weak coordination.     

An electrical potential in the access channel of catalases enhances catalysis

Substrate H2O2 must gain access to the deeply buried active site of catalases through channels of 30-50 A in length. The most prominent or main channel approaches the active site perpendicular to the plane of the heme and contains a number of residues that are conserved in all catalases. Changes in Val169, 8 A from the heme in catalase HPII from Escherichia coli, introducing smaller, larger or polar side chains reduces the catalase activity. Changes in Asp181, 12 A from the heme, reduces activity by up to 90% if the negatively charged side chain is removed when Ala, Gln, Ser, Asn, or Ile are the substituted residues. Only the D181E variant retains wild type activity. Determination of the crystal structures of the Glu181, Ala181, Ser181, and Gln181 variants of HPII reveals lower water occupancy in the main channel of the less active variants, particularly at the position forming the sixth ligand to the heme iron and in the hydrophobic, constricted region adjacent to Val169. It is proposed that an electrical potential exists between the negatively charged aspartate (or glutamate) side chain at position 181 and the positively charged heme iron 12 A distant. The potential field acts upon the electrical dipoles of water generating a common orientation that favors hydrogen bond formation and promotes interaction with the heme iron. Substrate hydrogen peroxide would be affected similarly and would enter the active site oriented optimally for interaction with active site residues.     

Substrate flow in catalases deduced from the crystal structures of active site variants of HPII from Escherichia coli.

The active site of heme catalases is buried deep inside a structurally highly conserved homotetramer. Channels leading to the active site have been identified as potential routes for substrate flow and product release, although evidence in support of this model is limited. To investigate further the role of protein structure and molecular channels in catalysis, the crystal structures of four active site variants of catalase HPII from Escherichia coli (His128Ala, His128Asn, Asn201Ala, and Asn201His) have been determined at approximately 2.0-A resolution. The solvent organization shows major rearrangements with respect to native HPII, not only in the vicinity of the replaced residues but also in the main molecular channel leading to the heme distal pocket. In the two inactive His128 variants, continuous chains of hydrogen bonded water molecules extend from the molecular surface to the heme distal pocket filling the main channel. The differences in continuity of solvent molecules between the native and variant structures illustrate how sensitive the solvent matrix is to subtle changes in structure. It is hypothesized that the slightly larger H(2)O(2) passing through the channel of the native enzyme will promote the formation of a continuous chain of solvent and peroxide. The structure of the His128Asn variant complexed with hydrogen peroxide has also been determined at 2.3-A resolution, revealing the existence of hydrogen peroxide binding sites both in the heme distal pocket and in the main channel. Unexpectedly, the largest changes in protein structure resulting from peroxide binding are clustered on the heme proximal side and mainly involve residues in only two subunits, leading to a departure from the 222-point group symmetry of the native enzyme. An active role for channels in the selective flow of substrates through the catalase molecule is proposed as an integral feature of the catalytic mechanism. The Asn201His variant of HPII was found to contain unoxidized heme b in combination with the proximal side His-Tyr bond suggesting that the mechanistic pathways of the two reactions can be uncoupled.     

Catalase HPII from Escherichia coli exhibits enhanced resistance to denaturation

Catalase HPII from Escherichia coli is a homotetramer of 753 residue subunits. The multimer displays a number of unusual structural features, including interwoven subunits and a covalent bond between Tyr415 and His392, that would contribute to its rigidity and stability. As the temperature of a solution of HPII in 50 mM potassium phosphate buffer (pH 7) is raised from 50 to 92 degrees C, the enzyme begins to lose activity at 78 degrees C and 50% inactivation has occurred at 83 degrees C. The inactivation is accompanied by absorbance changes at 280 and 407 nm and by changes in the CD spectrum consistent with small changes in secondary structure. The subunits in the dimer structure remain associated at 95 degrees C and show a significant level of dissociation only at 100 degrees C. The exceptional stability of the dimer association is consistent with the interwoven nature of the subunits and provides an explanation for the resistance to inactivation of the enzyme. For comparison, catalase-peroxidase HPI of E. coli and bovine liver catalase are 50% inactivated at 53 and 56 degrees C, respectively. In 5.6 M urea, HPII exhibits a coincidence of inactivation, CD spectral change, and dissociation of the dimer structure with a midpoint of 65 degrees C. The inactive mutant variants of HPII which fold poorly during synthesis and which lack the Tyr-His covalent bond undergo spectral changes in the 78 to 84 degrees C range, revealing that the extra covalent linkage is not important in the enhanced resistance to denaturation and that problems in the folding pathway do not affect the ultimate stability of the folded structure.     

Catalysis of proline-directed protein phosphorylation by peptidyl-prolyl cis/trans isomerases

Proline-directed protein phosphorylation was shown to depend on the capacity of the targeted Ser(Thr)-Pro bond to exhibit conformational polymorphism. The cis/trans isomer specificity underlying ERK2-catalyzed phosphate transfer leads to a complete discrimination of the cis Ser(Thr)-Pro conformer of oligopeptide substrates. We investigated in vitro the ERK2-catalyzed phosphorylation of Aspergillus oryzae RNase T1 containing two Ser-Pro bonds both of which share high stabilization energy in their respective native state conformation, the cis Ser54-Pro and the trans Ser72-Pro moiety. Despite trans isomer specificity of ERK2, a doubly phosphorylated RNase T1 was found as the final reaction product. Similarly, the RNase T1 S54G/P55N and RNase T1 P73V variants, which retain the prolyl bond conformations of the RNase T1-wt, were both monophosphorylated with a catalytic efficiency kcat/KM of 425 M(-1) s(-1) and 1228 M(-1) s(-1), respectively. However, initial phosphorylation rates did not depend linearly on the ERK2 concentration. The phosphorylation rate of the resulting plateau region at high ERK2 concentrations can be increased up to threefold for the RNase T1 P73V variant in the presence of the peptidyl-prolyl cis/trans isomerase Cyclophilin 18, indicating a conformational interconversion as the rate limiting step in the catalyzed phosphate group transfer. Using peptidyl-prolyl cis/trans isomerases with different substrate specificity, we identified a native state conformational equilibrium of the Ser54-Pro bond with the minor trans Ser54-Pro bond as the phosphorylation-sensitive moiety. This technique can therefore be used for a determination of the ratio and the interconversion rates of prolyl bond isomers in the native state of proteins.     

Coupling between trans/cis proline isomerization and protein stability in staphylococcal nuclease.

The nucleases A produced by two strains of Staphylococcus aureus, which have different stabilities, differ only in the identity of the single amino acid at residue 124. The nuclease from the Foggi strain of S. aureus (by convention nuclease WT), which contains His124, is 1.9 kcal.mol-1 less stable (at pH 5.5 and 20 degrees C) than the nuclease from the V8 strain (by convention nuclease H124L), which contains Leu124. In addition, the population of the trans conformer at the Lys116-Pro117 peptide bond, as observed by NMR spectroscopy, is different for the two variants: about 15% for nuclease WT and 9% for nuclease H124L. In order to improve our understanding of the origin of these differences, we compared the properties of WT and H124L with those of the H124A and H124I variants. We discovered a correlation between effects of different residues at this position on protein stability and on stabilization of the cis configuration of the Lys116-Pro117 peptide bond. In terms of free energy, approximately 17% of the increase in protein stability manifests itself as stabilization of the cis configuration at Lys116-Pro117. This result implies that the differences in stability arise mainly from structural differences between the cis configurational isomers at Pro117 of the different variants at residue 124. We solved the X-ray structure of the cis form of the most stable variant, H124L, and compared it with the published high-resolution X-ray structure of the cis form of the most stable variant, WT (Hynes TR, Fox RO, 1991, Proteins Struct Funct Genet 10:92-105). The two structures are identical within experimental error, except for the side chain at residue 124, which is exposed in the models of both variants. Thus, the increased stability and changes in the trans/cis equilibrium of the Lys116-Pro117 peptide bond observed in H124L relative to WT are due to subtle structural changes that are not observed by current structure determination technique. Residue 124 is located in a helix. However, the stability changes are too large and follow the wrong order of stability to be explained simply by differences in helical propensity. A second site of conformational heterogeneity in native nuclease is found at the His46-Pro47 peptide bond, which is approximately 80% trans in both WT and H124L. Because proline to glycine substitutions at either residue 47 or 117 remove the structural heterogeneity at that position and increase protein stability, we determined the X-ray structures of H124L + P117G and H124L + P47G + P117G and the kinetic parameters of H124L, H124L + P47G, H124L + P117G, and H124L + P47G + P117G. The individual P117G and P47G mutations cause decreases in nuclease activity, with kcat affected more than Km, and their effects are additive. The P117G mutation in nuclease H124L leads to the same local conformational rearrangement described for the P117G mutant of WT (Hynes TR, Hodel A, Fox RO, 1994, Biochemistry 33:5021-5030). In both P117G mutants, the loop formed by residues 112-117 is located closer to the adjacent loop formed by residues 77-85, and residues 115-118 adopt a type I' beta-turn conformation with the Lys116-Gly117 peptide bond in the trans configuration, as compared with the parent protein in which these residues have a typeVIa beta-turn conformation with the Lys116-Pro117 peptide bond in the cis configuration. Addition of the P47G mutation appears not to cause any additional structural changes. However, the electron density for part of the loop containing this peptide bond was not strong enough to be interpreted.     

Characterization of a large subunit catalase truncated by proteolytic cleavage

The large subunit catalase HPII from Escherichia coli can be truncated by proteolysis to a structure similar to small subunit catalases. Mass spectrometry analysis indicates that there is some heterogeneity in the precise cleavage sites, but approximately 74 N-terminal residues, 189 C-terminal residues, and a 9-11-residue internal fragment, including residues 298-308, are removed. Crystal structure refinement at 2.8 A reveals that the tertiary and quaternary structure of the native enzyme is retained with only very subtle changes despite the loss of 36% of the sequence. The truncated variant exhibits a 1.8 times faster turnover rate and enhanced sensitivity to high concentrations of H(2)O(2), consistent with easier access of the substrate to the active site. In addition, the truncated variant is more sensitive to inhibition, particularly by reagents such as aminotriazole and azide which are larger than substrate H(2)O(2). The main channel leading to the heme cavity is largely unaffected by the truncation, but the lateral channel is shortened and its entrance widened by removal of the C-terminal domain, providing an explanation for easier access to the active site. Opening of the entrance to the lateral channel also opens the putative NADPH binding site, but NADPH binding could not be demonstrated. Despite the lack of bound NADPH, the compound I species of both native and truncated HPII are reduced back to the resting state with compound II being evident in the absorbance spectrum only of the heme b-containing H392A variant.     

Mutagenic studies on histidine 98 of methylglyoxal synthase: effects on mechanism and conformational change

Two detailed mechanisms [Marks et al. (2001) Biochemistry 40, 6805] have been proposed to explain the activity of methylglyoxal synthase (MGS), a homohexameric allosterically regulated enzyme that catalyzes the elimination of phosphate from DHAP to form enol pyruvaldehyde. This enol then tautomerizes to methylglyoxal in solution. In one of these mechanisms His 98 plays an obligate role in the transfer of a proton from the O(3) oxygen of DHAP to the O2 oxygen. To test this hypothesized mechanism, the variants H98N and H98Q were expressed and purified. Relative to the wild-type enzyme, the H98N variant shows a 50-fold decrease in k(cat) with all other kinetic parameters (i.e., K(m), K(PGA), etc.) being nearly the same. By contrast, the apparent catalytic rate for the H98Q variant is 250-fold lower than that of the wild-type enzyme. Inorganic phosphate acts as a competitive inhibitor (with a K(i) of 15 microM) rather than as an allosteric-type inhibitor as it does in the wild-type enzyme, and the competitive inhibitor phosphoglyolate (PGA) acts as an activator of this variant. These facts are explained by a shift in the conformational equilibrium toward an "inactive" state. When activation by PGA is accounted for, the catalytic rate for the "active" state of H98Q is estimated to be only 6-fold less than that of the wild-type enzyme, and thus His 98 is not essential for catalysis. Primary deuterium isotope effect data were measured for the wild-type enzyme and the two variants. At pH 7.0, the (D)V isotope effect (1.5) and the absence of a (D)(V/K) isotope effect for the wild-type enzyme suggest that the rate for the isotopically sensitive step is partially rate limiting but greater than the rate of substrate dissociation. Both the (D)V (2.0) and (D)(V/K) (3.4) isotope effects were more pronounced in the H98N variant, while the H98Q variant displayed an unusual inverse (D)V (0.8) isotope effect and a normal (D)(V/K) (1.5) isotope effect. The X-ray crystal structures of PGA bound to the H98Q and H98N variants were both determined to a resolution of 2.2 A. These mutations had little effect on the overall structure. However, the pattern of hydrogen bonding in the active site suggests an explanation as to how in the wild-type and H98N mutated enzymes the "inactive to active" equilibrium lies toward the active state, while with the H98Q mutated enzyme the equilibrium lies toward the inactive state. Thus, the role of His 98 appears to be more as a regulator of the enzyme's conformation rather than as a critical contributor to the catalytic mechanism.     

Structural basis for the inhibition of the biosynthesis of biotin by the antibiotic amiclenomycin

The antibiotic amiclenomycin blocks the biosynthesis of biotin by inhibiting the pyridoxal-phosphate-dependent enzyme diaminopelargonic acid synthase. Inactivation of the enzyme is stereoselective, i.e. the cis isomer of amiclenomycin is a potent inhibitor, whereas the trans isomer is much less reactive. The crystal structure of the complex of the holoenzyme and amiclenomycin at 1.8 A resolution reveals that the internal aldimine linkage between the cofactor and the side chain of the catalytic residue Lys-274 is broken. Instead, a covalent bond is formed between the 4-amino nitrogen of amiclenomycin and the C4' carbon atom of pyridoxal-phosphate. The electron density for the bound inhibitor suggests that aromatization of the cyclohexadiene ring has occurred upon formation of the covalent adduct. This process could be initiated by proton abstraction at the C4 carbon atom of the cyclohexadiene ring, possibly by the proximal side chain of Lys-274, leading to the tautomer Schiff base followed by the removal of the second allylic hydrogen. The carboxyl tail of the amiclenomycin moiety forms a salt link to the conserved residue Arg-391 in the substrate-binding site. Modeling suggests steric hindrance at the active site as the determinant of the weak inhibiting potency of the trans isomer.     

Influence of the covalent heme-protein bonds on the redox thermodynamics of human myeloperoxidase

Myeloperoxidase (MPO) is the most abundant neutrophil enzyme and catalyzes predominantly the two-electron oxidation of ubiquitous chloride to generate the potent bleaching hypochlorous acid, thus contributing to pathogen killing as well as inflammatory diseases. Its catalytic properties are closely related with unique posttranslational modifications of its prosthetic group. In MPO, modified heme b is covalently bound to the protein via two ester linkages and one sulfonium ion linkage with a strong impact on its (electronic) structure and biophysical and chemical properties. Here, the thermodynamics of the one-electron reduction of the ferric heme in wild-type recombinant MPO and variants with disrupted heme-protein bonds (M243V, E242Q, and D94V) have been investigated by thin-layer spectroelectrochemistry. It turns out that neither the oligomeric structure nor the N-terminal extension in recombinant MPO modifies the peculiar positive reduction potential (E°' = 0.001 V at 25 °C and pH 7.0) or the enthalpy or entropy of the Fe(III) to Fe(II) reduction. By contrast, upon disruption of the MPO-typical sulfonium ion linkage, the reduction potential is significantly lower (-0.182 V). The M243V mutant has an enthalpically stabilized ferric state, whereas its ferrous form is entropically favored because of the loss of rigidity of the distal H-bonding network. Exchange of an adjacent ester bond (E242Q) induced similar but less pronounced effects (E°' = -0.094 V), whereas in the D94V variant (E°' = -0.060 V), formation of the ferrous state is entropically disfavored. These findings are discussed with respect to the chlorination and bromination activity of the wild-type protein and the mutants.     

Role of the lateral channel in catalase HPII of Escherichia coli

The heme-containing catalase HPII of Escherichia coli consists of a homotetramer in which each subunit contains a core region with the highly conserved catalase tertiary structure, to which are appended N- and C-terminal extensions making it the largest known catalase. HPII does not bind NADPH, a cofactor often found in catalases. In HPII, residues 585-590 of the C-terminal extension protrude into the pocket corresponding to the NADPH binding site in the bovine liver catalase. Despite this difference, residues that define the NADPH pocket in the bovine enzyme appear to be well preserved in HPII. Only two residues that interact ionically with NADPH in the bovine enzyme (Asp212 and His304) differ in HPII (Glu270 and Glu362), but their mutation to the bovine sequence did not promote nucleotide binding. The active-site heme groups are deeply buried inside the molecular structure requiring the movement of substrate and products through long channels. One potential channel is about 30 A in length, approaches the heme active site laterally, and is structurally related to the branched channel associated with the NADPH binding pocket in catalases that bind the dinucleotide. In HPII, the upper branch of this channel is interrupted by the presence of Arg260 ionically bound to Glu270. When Arg260 is replaced by alanine, there is a threefold increase in the catalytic activity of the enzyme. Inhibitors of HPII, including azide, cyanide, various sulfhydryl reagents, and alkylhydroxylamine derivatives, are effective at lower concentration on the Ala260 mutant enzyme compared to the wild-type enzyme. The crystal structure of the Ala260 mutant variant of HPII, determined at 2.3 A resolution, revealed a number of local structural changes resulting in the opening of a second branch in the lateral channel, which appears to be used by inhibitors for access to the active site, either as an inlet channel for substrate or an exhaust channel for reaction products.     

Inactivation of the Heme Degrading Enzyme IsdI by an Active Site Substitution That Diminishes Heme Ruffling

IsdG and IsdI are paralogous heme degrading enzymes from the bacterium Staphylococcus aureus. Heme bound by these enzymes is extensively ruffled such that the meso-carbons at the sites of oxidation are distorted toward bound oxygen. In contrast, the canonical heme oxygenase family degrades heme that is bound with minimal distortion. Trp-66 is a conserved heme pocket residue in IsdI implicated in heme ruffling. IsdI variants with Trp-66 replaced with residues having less bulky aromatic and alkyl side chains were characterized with respect to catalytic activity, heme ruffling, and electrochemical properties. The heme degradation activity of the W66Y and W66F variants was approximately half that of the wild-type enzyme, whereas the W66L and W66A variants were inactive. A crystal structure and NMR spectroscopic analysis of the W66Y variant reveals that heme binds to this enzyme with less heme ruffling than observed for wild-type IsdI. The reduction potential of this variant (−96 ± 7 mV versus standard hydrogen electrode) is similar to that of wild-type IsdI (−89 ± 7 mV), so we attribute the diminished activity of this variant to the diminished heme ruffling observed for heme bound to this enzyme and conclude that Trp-66 is required for optimal catalytic activity.     

Modulation of heme orientation and binding by a single residue in catalase HPII of Escherichia coli

Heme-containing catalases have been extensively studied, revealing the roles of many residues, the existence of two heme orientations, flipped 180° relative to one another along the propionate-vinyl axis, and the presence of both heme b and heme d. The focus of this report is a residue, situated adjacent to the vinyl groups of the heme at the entrance of the lateral channel, with an unusual main chain geometry that is conserved in all catalase structures so far determined. In Escherichia coli catalase HPII, the residue is Ile274, and replacing it with Gly, Ala, and Val, found at the same location in other catalases, results in a reduction in catalytic efficiency, a reduced intensity of the Soret absorbance band, and a mixture of heme orientations and species. The reduced turnover rates and higher H(2)O(2) concentrations required to attain equivalent reaction velocities are explained in terms of less efficient containment of substrate H(2)O(2) in the heme cavity arising from easier escape through the more open entrance to the lateral channel created by the smaller side chains of Gly and Ala. Inserting a Cys at position 274 resulted in the heme being covalently linked to the protein through a Cys-vinyl bond that is hypersensitive to X-ray irradiation being largely degraded within seconds of exposure to the X-ray beam. Two heme orientations, flipped along the propionate-vinyl axis, are found in the Ala, Val, and Cys variants.     

1H NMR studies of the effect of mutation at Valine45 on heme microenvironment of cytochrome b5

1D and 2D (1)H NMR were employed to probe the effects on the heme microenvironment of cytochrome b(5) caused by the mutation from Val45 to Tyr45, His45 and Glu45. Compared with wild type (WT) cytochrome b(5), in all mutants the heme ring are CCW rotated relative to the imidazole planes of axial ligands and the angles beta between two axial ligand imidazole planes are not changed, being in agreement with the temperature dependence of the shifts of the heme protons. The ratios of heme isomers (major to minor) are smaller than that in WT. The 4-vinyl group of the heme in V45Y assumes cis-orientation, being similar to that of WT, while in V45E and V45H, both cis and trans orientation are found. The relationships between the structure and biological function of the mutants are discussed in terms of the geometry of heme and axial ligands, the hydrophobicity of heme pocket and the electrostatic potential of the heme-exposed area.     

Asp-225 and glu-375 in autocatalytic attachment of the prosthetic heme group of lactoperoxidase.

The heme in lactoperoxidase is attached to the protein by ester bonds between the heme 1- and 5-methyl groups and Glu-375 and Asp-275, respectively. To investigate the cross-linking process, we have examined the D225E, E375D, and D225E/E375D mutants of bovine lactoperoxidase. The heme in the E375D mutant is only partially covalently bound, but exposure to H(2)O(2) results in complete covalent binding and a fully active protein. Digestion of this mutant shows that the heme is primarily bound through its 5-methyl group. Excess H(2)O(2) increases the proportion of the doubly linked species without augmenting enzyme activity. The D225E mutant has little covalently bound heme and a much lower activity, neither of which are significantly increased by the addition of heme and H(2)O(2). The heme is linked to this protein through a single bond to the 1-methyl group. The D225E/E375D mutant has no covalently bound heme and no activity. A small amount of iron 1-hydroxymethylprotoporphyrin IX is obtained from the wild-type enzyme along with the predominant dihydroxylated derivative. The results establish that a single covalent link suffices to achieve maximum catalytic activity and suggest that the 5-hydroxymethyl bond may form before the 1-hydroxymethyl bond.     

Catalytic role of the active site histidine of porcine pancreatic phospholipase A2 probed by the variants H48Q, H48N and H48K.

The catalytic contribution of His48 in the active site of porcine pancreatic phospholipase A2 was examined using site-directed mutagenesis. Replacement of His48 by lysine (H48K) gives rise to a protein having a distorted lipid binding pocket. Activity of this variant drops below the detection limit which is 10(7)-fold lower than that of the wild-type enzyme. On the other hand, the presence of glutamine (H48Q) or asparagine (H48N) at this position does not affect the structural integrity of the enzyme as can be derived from the preserved lipid binding properties of these variants. However, the substitutions H48Q and H48N strongly reduce the turnover number, i.e. by a factor of 10(5). Residual activity is totally lost after addition of a competitive inhibitor. We conclude that proper lipid binding on its own accelerates ester bond hydrolysis by a factor of 10(2). With the selected variants, we were also able to dissect the contribution of the hydrogen bond between Asp99 and His48 on conformational stability, being 5.2 kJ/mol. Another hydrogen bond with His48 is formed when the competitive inhibitor (R)-2-dodecanoylamino-hexanol-1-phosphoglycol interacts with the enzyme. Its contribution to binding of the inhibitor in the presence of an interface was found to be 5.7 kJ/mol.     

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.     

Stabilization of a strained protein loop conformation through protein engineering.

Staphylococcal nuclease is found in two folded conformations that differ in the isomerization of the Lys 116-Pro 117 peptide bond, resulting in two different conformations of the residue 112-117 loop. The cis form is favored over the trans with an occupancy of 90%. Previous mutagenesis studies have shown that when Lys 116 is replaced by glycine, a trans conformation is stabilized relative to the cis conformation by the release of steric strain in the trans form. However, when Lys 116 is replaced with alanine, the resulting variant protein is identical to the wild-type protein in its structure and in the dominance of the cis configuration. The results of these studies suggested that any nuclease variant with a non-glycine residue at position 116 should also favor the cis form because of steric requirements of the beta-carbon at this position. In this report, we present a structural analysis of four nuclease variants with substitutions at position 116. Two variants, K116E and K116M, follow the "beta-carbon" hypothesis by favoring the cis form. Furthermore, the crystal structure of K116E is nearly identical to that of the wild-type protein. Two additional variants, K116D and K116N, provide exceptions to this simple "beta-carbon" rule in that the trans conformation is stabilized relative to the cis configuration by these substitutions. Crystallographic data indicate that this stabilization is effected through the addition of tertiary interactions between the side chain of position 116 with the surrounding protein and water structure. The detailed trans conformation of the K116D variant appears to be similar to the trans conformation observed in the K116G variant, suggesting that these two mutations stabilize the same conformation but through different mechanisms.     

Inhibition of beta-lactamase by clavulanate. Trapped intermediates in cryocrystallographic studies.

Crystallographic studies of the complex between beta-lactamase and clavulanate reveal a structure of two acyl-enzymes with covalent bonds at the active site Ser70, representing two different stages of inhibitor degradation alternately occupying the active site. Models that are consistent with biochemical data are derived from the electron density map and refined at 2.2 A resolution: cis enamine, in which the carboxylate group of the clavulanate molecule makes a salt bridge with Lys234 of beta-lactamase; decarboxylated trans enamine, which is oriented away from Lys234. For both acyl-enzymes, the carbonyl oxygen atom of the ester group occupies the oxyanion hole in a manner similar to that found in inhibitor binding to serine proteases. Whereas the oxygen atom in the trans product is optimally positioned in the oxyanion hole, that of the cis product clashes with the main-chain nitrogen atom of Ser70 and the beta-carbon atom of the adjacent Ala69. In contrast to cis to trans isomerization in solution that relieves the steric strain inherent in a cis double bond, at the enzyme-inhibitor interface two additional factors play an important role. The salt bridge enhances the stability of the cis product, while the steric strain introduced by the short contacts with the protein reduces its stability.