Active site properties of monomeric triosephosphate isomerase (monoTIM) as deduced from mutational and structural studies.

MonoTIM is a stable monomeric variant of the dimeric trypanosomal enzyme triose phosphate isomerase (TIM) with less, but significant, catalytic activity. It is known that in TIM, three residues, Lys 13 (loop 1), His 95 (loop 4), and Glu 167 (loop 6) are the crucial catalytic residues. In the wild-type TIM dimer, loop 1 and loop 4 are very rigid because of tight interactions with residues of the other subunit. Previous structural studies indicate that Lys 13 and His 95 have much increased conformational flexibility in monoTIM. Using site-directed mutagenesis, it is shown here that Lys 13 and His 95 are nevertheless essential for optimal catalysis by monoTIM: monoTIM-K13A is completely inactive, although it can still bind substrate analogues, and monoTIM-H95A is 50 times less active. The best inhibitors of wild-type TIM are phosphoglycolohydroxamate (PGH) and 2-phosphoglycolate (2PG), with KI values of 8 microM and 26 microM, respectively. The affinity of the monoTIM active site for PGH has been reduced approximately 60-fold, whereas for 2PG, only a twofold weakening of affinity is observed. The mode of binding, as determined by protein crystallographic analysis of these substrate analogues, shows that, in particular, 2PG interacts with Lys 13 and His 95 in a way similar but not identical to that observed for the wild-type enzyme. This crystallographic analysis also shows that Glu 167 has the same interactions with the substrate analogues as in the wild type. The data presented suggest that, despite the absence of the second subunit, monoTIM catalyzes the interconversion of D-glyceraldehyde-3-phosphate and dihydroxyacetone phosphate via the same mechanism as in the wild type.     

The planar conformation of a strained proline ring: a QM/MM study

QM and QM/MM energy calculations have been carried out on an atomic resolution structure of liganded triosephosphate isomerase (TIM) that has an active site proline (Pro168) in a planar conformation. The origin of the planarity of this proline has been identified. Steric interactions between the atoms of the proline ring and a tyrosine ring (Tyr166) on one side of the proline prevent the ring from adopting the up pucker (chi1 is approximately -30 degrees), while the side chain of a nearby alanine (Ala171) forbids the down pucker (chi1 is approximately +30 degrees). To obtain a proline conformation that is in agreement with the experimentally observed planar state, a quantum system of sufficient size is required and should at least include the nearby side chains of Tyr166, Ala171, and Glu129 to provide enough stabilization. It is argued that the current force fields for structure optimization do not describe strained protein fragments correctly. The proline is part of a catalytic loop that closes upon ligand binding. Comparison of the proline conformation in different TIM X-ray structures, indicates that in the closed conformation of TIM the proline is planar or nearly planar, while in the open conformation it is down puckered. This suggests that the planarity possibly plays a role in the overall catalytic cycle of TIM, presumable acting as a reservoir of energy that becomes available upon loop opening.     

Crystal structure of triosephosphate isomerase complexed with 2-phosphoglycolate at 0.83-A resolution

The atomic resolution structure of Leishmania mexicana triosephosphate isomerase complexed with 2-phosphoglycolate shows that this transition state analogue is bound in two conformations. Also for the side chain of the catalytic glutamate, Glu(167), two conformations are observed. In both conformations, a very short hydrogen bond exists between the carboxylate group of the ligand and the catalytic glutamate. The distance between O11 of PGA and Oepsilon2 of Glu(167) is 2.61 and 2.55 A for the major and minor conformations, respectively. In either conformation, Oepsilon1 of Glu(167) is hydrogen-bonded to a water network connecting the side chain with bulk solvent. This network also occurs in two mutually exclusive arrangements. Despite the structural disorder in the active site, the C termini of the beta strands that construct the active site display the least anisotropy compared with the rest of the protein. The loops following these beta strands display various degrees of anisotropy, with the tip of the dimer interface loop 3 having very low anisotropy and the C-terminal region of the active site loop 6 having the highest anisotropy. The pyrrolidine ring of Pro(168) at the N-terminal region of loop 6 is in a strained planar conformation to facilitate loop opening and product release.     

Atomic resolution crystallography of a complex of triosephosphate isomerase with a reaction‐intermediate analog: New insight in the proton transfer reaction mechanism

Enzymes achieve their catalytic proficiency by precisely positioning the substrate and catalytic residues with respect to each other. Atomic resolution crystallography is an excellent tool to study the important details of these geometric active‐site features. Here, we have investigated the reaction mechanism of triosephosphate isomerase (TIM) using atomic resolution crystallographic studies at 0.82‐Å resolution of leishmanial TIM complexed with the well‐studied reaction‐intermediate analog phosphoglycolohydroxamate (PGH). Remaining unresolved aspects of the reaction mechanism of TIM such as the protonation state of the first reaction intermediate and the properties of the hydrogen‐bonding interactions in the active site are being addressed. The hydroxamate moiety of PGH interacts via unusually short hydrogen bonds of its N1? O1 moiety with the carboxylate group of the catalytic glutamate (Glu167), for example, the distance of N1(PGH)‐OE2(Glu167) is 2.69 ± 0.01 Å and the distance of O1(PGH)‐OE1(Glu167) is 2.60 ± 0.01 Å. Structural comparisons show that the side chain of the catalytic base (Glu167) can move during the reaction cycle in a small cavity, located above the hydroxamate plane. The structure analysis suggests that the hydroxamate moiety of PGH is negatively charged. Therefore, the bound PGH mimics the negatively charged enediolate intermediate, which is formed immediately after the initial proton abstraction from DHAP by the catalytic glutamate. The new findings are discussed in the context of the current knowledge of the TIM reaction mechanism. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Dynamic requirements for a functional protein hinge

The enzyme triosephosphate isomerase (TIM) is a model of catalytic efficiency. The 11 residue loop 6 at the TIM active site plays a major role in this enzymatic prowess. The loop moves between open and closed states, which facilitate substrate access and catalysis, respectively. The N and C-terminal hinges of loop 6 control this motion. Here, we detail flexibility requirements for hinges in a comparative solution NMR study of wild-type (WT) TIM and a quintuple mutant (PGG/GGG). The latter contained glycine substitutions in the N-terminal hinge at Val167 and Trp168, which follow the essential Pro166, and in the C-terminal hinge at Lys174, Thr175, and Ala176. Previous work demonstrated that PGG/GGG has a tenfold higher Km value and 10(3)-fold reduced k(cat) relative to WT with either d-glyceraldehyde 3-phosphate or dihyrdroxyacetone phosphate as substrate. Our NMR results explain this in terms of altered loop-6 dynamics in PGG/GGG. In the mutant, loop 6 exhibits conformational heterogeneity with corresponding motional rates <750 s(-1) that are an order of magnitude slower than the natural WT loop 6 motion. At the same time, nanosecond timescale motions of loop 6 are greatly enhanced in the mutant relative to WT. These differences from WT behavior occur in both apo PGG/GGG and in the form bound to the reaction-intermediate analog, 2-phosphoglycolate (2-PGA). In addition, as indicated by 1H, 15N and 13CO chemical-shifts, the glycine substitutions diminished the enzyme's response to ligand, and induced structural perturbations in apo and 2-PGA-bound forms of TIM that are atypical of WT. These data show that PGG/GGG exists in multiple conformations that are not fully competent for ligand binding or catalysis. These experiments elucidate an important principle of catalytic hinge design in proteins: structural rigidity is essential for focused motional freedom of active-site loops.     

Entropy effects on protein hinges: the reaction catalyzed by triosephosphate isomerase

Many proteins utilize segmental motions to catalyze a specific reaction. The Omega loop of triosephosphate isomerase (TIM) is important for preventing the loss of the reactive enediol(ate) intermediate. The loop opens and closes even in the absence of the ligand, and the loop itself does not change conformation during movement. The conformational changes are localized to two hinges at the loop termini. Glycine is never observed in native TIM hinge sequences. In this paper, the hypothesis that limited access to conformational space is a requirement for protein hinges involved in catalysis was tested. The N-terminal hinge was mutated to P166/V167G/W168G (PGG), and the C-terminal hinge was mutated to K174G/T175G/A176G (GGG) in chicken TIM. The single-hinge mutants PGG and GGG had k(cat) values 200-fold lower than that of the wild type and K(m) values 10-fold higher. The k(cat) of double-hinge mutant P166/V167G/W168G/K174G/T175G/A176G was reduced 2500-fold; the K(m) was 10-fold higher. A combination of primary kinetic isotope effect measurements, isothermal calorimetric measurements, and (31)P NMR spectroscopic titration with the inhibitor 2-phosphoglycolate revealed that the mutants have a different ligand-binding mode than that of the wild-type enzyme. The predominant conformations of the mutants even in the presence of the inhibitor are loop-open conformations. In conclusion, mutation of the hinge residues to glycine resulted in the sampling of many more hinge conformations with the consequence that the population of the active-closed conformation is reduced. This reduced population results in a reduced catalytic activity.     

X-ray structure analyses of photosynthetic reaction center variants from Rhodobacter sphaeroides: structural changes induced by point mutations at position L209 modulate electron and proton transfer

The structures of the reaction center variants Pro L209 --> Tyr, Pro L209 --> Phe, and Pro L209 --> Glu from the photosynthetic purple bacterium Rhodobacter sphaeroides have been determined by X-ray crystallography to 2.6-2.8 A resolution. These variants were constructed to interrupt a chain of tightly bound water molecules that was assumed to facilitate proton transfer from the cytoplasm to the secondary quinone Q(B) [Baciou, L., and Michel, H. (1995) Biochemistry 34, 7967-7972]. However, the amino acid exchanges Pro L209 --> Tyr and Pro L209 --> Phe do not interrupt the water chain. Both aromatic side chains are oriented away from this water chain and interact with three surrounding polar side chains (Asp L213, Thr L226, and Glu H173) which are displaced by up to 2.6 A. The conformational changes induced by the bulky aromatic rings of Tyr L209 and Phe L209 lead to unexpected displacements of Q(B) compared to the wild-type protein. In the structure of the Pro L209 --> Tyr variant, Q(B) is shifted by approximately 4 A and is now located at a position similar to that reported for the wild-type reaction center after illumination [Stowell, M. H. B., et al. (1997) Science 276, 812-816]. In the Pro L209 --> Phe variant, the electron density map reveals an intermediate Q(B) position between the binding sites of the wild-type protein in the dark and the Pro L209 --> Tyr protein. In the Pro L209 --> Glu reaction center, the carboxylic side chain of Glu L209 is located within the water chain, and the binding site of Q(B) remains unchanged compared to the wild-type structure.     

Mirroring perfection: the structure of methylglyoxal synthase complexed with the competitive inhibitor 2-phosphoglycolate

The crystal structure of the transition-state analogue 2-phosphoglycolate (2PG) bound to methylglyoxal synthase (MGS) is presented at a resolution of 2.0 A. This structure is very similar to the previously determined structure of MGS complexed to formate and phosphate. Since 2PG is a competitive inhibitor of both MGS and triosephosphate isomerase (TIM), the carboxylate groups of each bound 2PG from this structure and the structure of 2PG bound to TIM were used to align and compare the active sites despite differences in their protein folds. The distances between the functional groups of Asp 71, His 98, His 19, and the carboxylate oxygens of the 2PG molecule in MGS are similar to the corresponding distances between the functional groups of Glu 165, His 95, Lys 13, and the carboxylate oxygens of the 2PG molecule in TIM. However, these spatial relationships are enantiomorphic to each other. Consistent with the known stereochemical data, the catalytic base Asp 71 is positioned on the opposite face of the 2PG-carboxylate plane as Glu 165 of TIM. Both His 98 of MGS and His 95 of TIM are in the plane of the carboxylate of 2PG, suggesting that these two residues are homologous in function. While His 19 of MGS and Lys 13 of TIM appear on the opposite face of the 2PG carboxylate plane, their relative location to the 2PG molecule is quite different, suggesting that they probably have different functions. Most remarkably, unlike the coplanar structure found in the 2PG molecule bound to TIM, the torsion angle around the C1-C2 bond of 2PG bound to MGS brings the phosphoryl moiety out of the molecule's carboxylate plane, facilitating elimination. Further, the superimposition of this structure with the structure of MGS bound to formate and phosphate suggests a model for the enzyme bound to the first transition state.     

High resolution crystal structures of triosephosphate isomerase complexed with its suicide inhibitors: The conformational flexibility of the catalytic glutamate in its closed, liganded active site

The key residue of the active site of triosephosphate isomerase (TIM) is the catalytic glutamate, which is proposed to be important (i) as a catalytic base, for initiating the reaction, as well as (ii) for the subsequent proton shuttling steps. The structural properties of this glutamate in the liganded complex have been investigated by studying the high resolution crystal structures of typanosomal TIM, complexed with three suicide inhibitors: (S)-glycidol phosphate ((S)-GOP, at 0.99 Å resolution), (R)-glycidol phosphate, ((R)-GOP, at 1.08 Å resolution), and bromohydroxyacetone phosphate (BHAP, at 1.97 Å resolution). The structures show that in the (S)-GOP active site this catalytic glutamate is in the well characterized, competent conformation. However, an unusual side chain conformation is observed in the (R)-GOP and BHAP complexes. In addition, Glu97, salt bridged to the catalytic lysine in the competent active site, adopts an unusual side chain conformation in these two latter complexes. The higher chemical reactivity of (S)-GOP compared with (R)-GOP, as known from solution studies, can be understood: the structures indicate that in the case of (S)-GOP, Glu167 can attack the terminal carbon of the epoxide in a stereoelectronically favored, nearly linear O–C–O arrangement, but this is not possible for the (R)-GOP isomer. These structures confirm the previously proposed conformational flexibility of the catalytic glutamate in its closed, liganded state. The importance of this conformational flexibility for the proton shuttling steps in the TIM catalytic cycle, which is apparently achieved by a sliding motion of the side chain carboxylate group above the enediolate plane, is also discussed.     

A role for flexible loops in enzyme catalysis

Triosephosphate isomerase (TIM), glycerol 3-phosphate dehydrogenase, and orotidine 5'-monophosphate decarboxylase each use the binding energy from the interaction of phosphite dianion with a flexible phosphate gripper loop to activate a second, phosphodianion-truncated, substrate towards enzyme-catalyzed proton transfer, hydride transfer, and decarboxylation, respectively. Studies on TIM suggest that the most important general effect of loop closure over the substrate phosphodianion, and the associated conformational changes, is to extrude water from the enzyme active site. This should cause a decrease in the effective active-site dielectric constant, and an increase in transition state stabilization from enhanced electrostatic interactions with polar amino acid side chains. The most important specific effect of these conformational changes is to increase the basicity of the carboxylate side chain of the active site glutamate base by its placement in a 'hydrophobic cage'.     

Refined 1.83 A structure of trypanosomal triosephosphate isomerase crystallized in the presence of 2.4 M-ammonium sulphate. A comparison with the structure of the trypanosomal triosephosphate isomerase-glycerol-3-phosphate complex

Triosephosphate isomerase (TIM) is a dimeric glycolytic enzyme. TIM from Trypanosoma brucei brucei has been crystallized at pH 7.0 in 2.4 M-ammonium sulphate. The well-diffracting crystals have one dimer per asymmetric unit. The structure has been refined at 1.83 A resolution with an R-factor of 18.3% for all data between 6 A and 1.83 A (37,568 reflections). The model consists of 3778 protein atoms and 297 solvent atoms. Subunit 1 is involved in considerably more crystal contacts than subunit 2. Correlated with these differences in crystal packing is the observation that only in the active site of subunit 2 is a sulphate ion bound. Furthermore, significant differences with respect to structure and flexibility are observed in three loops near the active site. In particular, there is a 7 A positional difference of the tip of the flexible loop (loop 6) when comparing subunit 1 and subunit 2. Also, the neighbouring loops (loop 5 and loop 7) have significantly different conformations and flexibility. In subunit 1, loop 6 is in an "open" conformation, in subunit 2, loop 6 is in an "almost closed" conformation. Only in the presence of a phosphate-containing ligand, such as glycerol-3-phosphate, does loop 6 take up the "closed" conformation. Loop 6 and loop 7 (and also to some extent loop 5) are rather flexible in the almost closed conformation, but well defined in the open and closed conformations. The closing of loop 6 (167 to 180), as observed in the almost closed conformation, slightly changes the main-chain conformation of the catalytic glutamate, Glu167, leading to a change of the chi 1 angle of this residue from approximately -60 degrees to approximately 60 degrees and the weakening of the hydrogen bonds between its polar side-chain atoms and Ser96. In the closed conformation, in the presence of glycerol-3-phosphate, the main-chain atoms of Glu167 remain in the same position as in the almost closed conformation, but the side-chain has rotated around the CA-CB bond changing chi 1 from approximately 60 degrees to approximately -60 degrees. In this new position the hydrogen bonding to Ser96 is completely lost and also a water-mediated salt bridge between OE2(Glu167) and NE(Arg99) is lost. Comparison of the two independently refined subunits, showed that the root-mean-square deviation for all 249 CA atoms is 0.9 A; for the CA atoms of the beta-strands this is only 0.2 A. The average B-factor for all subunit 1 and subunit 2 atoms is 20 A2 and 25 A2, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

The hppA gene of Helicobacter pylori encodes the class C acid phosphatase precursor

Triosephosphate isomerase (TIM) has a conserved salt bridge 20 A away from both the active site and the dimer interface. In this study, four salt bridge mutants of Trypanosoma brucei brucei TIM were characterized. The folding and stability of the mutants are impaired compared to the wild-type enzyme. This salt bridge is part of a hydrogen bonding network which tethers the C-terminal beta7alpha7beta8alpha8 unit to the bulk of the protein. In the variants D227N, D227A, and R191S, this network is preserved, as can be deduced from the structure of the R191S variant. In the R191A variant, the side chain at position 191 cannot contribute to this network. Also the catalytic power of this variant is most affected.     

The structural basis of a conserved P2 threonine in canonical serine proteinase inhibitors

Bowman-Birk inhibitors (BBIs) are a well-studied family of canonical inhibitor proteins of serine proteinases. In nature, the active region of BBIs possesses a highly conserved Thr at the P2 position. The importance of this residue has been reemphasized by synthetic BBI reactive site loop proteinomimetics. In particular, this residue was exclusively identified for active chymotrypsin inhibitors selected from a BBI template-assisted combinatorial peptide library. A further kinetic analysis of 26 P2 variant peptides revealed that Thr provides both optimal binding affinity and optimal resistance against enzymatic turnover by chymotrypsin. Herein, we report the (1)H-NMR spectroscopic study of a 5-membered sub-set of these reactive site loop peptides representing a stepwise elimination of the Thr side-chain functionalities and inversion of its side-chain chirality. The P2 Thr variant adopts a three-dimensional structure that closely mimics the one of the corresponding region of the complete protein. This validates the use of this template for the investigation of structure-function relationships. While the overall backbone geometry is similar in all studied variants, conformational changes induced by the modification of the P2 side chain have now been identified and provide a rational explanation of the kinetically observed functional differences. Eliminating the gamma-methyl group has little structural effect, whereas the elimination of the gamma-oxygen atom or the inversion of the side-chain chirality results in characteristic changes to the intramolecular hydrogen bond network. We conclude that the transannular hydrogen bond between the P2 Thr side-chain hydroxyl and the P5' backbone amide is an important conformational constraint and directs the hydrophobic contact of the P2 Thr side chain with the enzyme surface in a functionally optimal geometry, both in the proteinomimetic and the native protein. In at least four canonical inhibitor protein families similar structural arrangements for a conserved P2 Thr have been observed, which suggests an analogous functional role. Substitutions at P2 of the proteinomimetic also affect the conformational balance between cis and trans isomers at a distant Pro-Pro motif (P3'-P4'). Presented with a mixture of cis/trans isomers chymotrypsin appears to interact preferably with the conformer that retains the cis-P3' Pro-trans-P4' Pro geometry found in the parent BBI protein.     

Protein engineering studies of A-chain loop 47-56 of Escherichia coli heat-labile enterotoxin point to a prominent role of this loop for cytotoxicity

Heat-labile enterotoxin (LT), produced by enterotoxigenic Escherichia coli, is a close relative of cholera toxin (CT). These two toxins share approximately 80% sequence identity, and consists of one 240-residue A chain and five 103-residue B subunits. The B pentamer is responsible for G_M1 receptor recognition, whereas the A subunit carries out an ADP-ribosylation of an arginine residue in the G protein, G_Sα, in the epithelial target cell. This paper explores the importance of specific amino acids in loop 47–56 of the A subunit. This loop was observed to be highly mobile in the inactive R7K mutant of the A subunit. The position of the loop in wild-type protein is such that it might require considerable reorganization during substrate binding and is likely to have a crucial role in substrate binding. Five single-site substitutions have been made in the LT-A subunit 47–56 loop to investigate its possible role in the enzymatic activity and toxicity of LT and CT. The wild-type residues Thr-50 and Val-53 were replaced either by a glycine or by a proline. The glycine substitutions were intended to increase the mobility of this active-site loop, and the proline substitutions were intended to decrease the mobility of this same loop by restricting the accessible conformational space. Under the hypothesis that mobility of the loop is important for catalysis, the glycine-substitution mutants T50G and V53G would be expected to exhibit activity equal to or greater than that of the wild-type A subunit, while the proline substitution mutants T50P and T53P would be less active. Cytotoxicity assays showed, however, that all four of these mutants were considerably less active than wild-type LT. These results lend support for assignment of a prominent role to loop 47–56 in catalysis by LT and CT.     

Crystal structures of triosephosphate isomerase from methicillin resistant Staphylococcus aureus MRSA252 provide structural insights into novel modes of ligand binding and unique conformations of catalytic loop

Staphylococcus aureus is one of the most dreaded pathogens worldwide and emergence of notorious antibiotic resistant strains have further exacerbated the present scenario. The glycolytic enzyme, triosephosphate isomerase (TIM) is one of the cell envelope proteins of the coccus and is involved in biofilm formation. It also plays an instrumental role in adherence and invasion of the bacteria into the host cell. To structurally characterize this important enzyme and analyze it's interaction with different inhibitors, substrate and transition state analogues, the present article describes several crystal structures of SaTIM alone and in complex with different ligands: glycerol-3-phosphate (G3P), glycerol-2-phosphate (G2P), 3-phosphoglyceric acid (3PG) and 2-phosphoglyceric acid (2PG). Unique conformations of the catalytic loop 6 (L6) has been observed in the different complexes. It is found to be in “almost closed” conformation in both subunits of the structure complexed to G3P. However L6 adopts the open conformation in presence of G2P and 2PG. The preference of the conformation of the catalytic loop can be correlated with the position of the phosphate group in the ligand. Novel modes of binding have been observed for G2P and 3PG for the very first time. The triose moiety is oriented away from the catalytic residues and occupies an entirely different position in some subunits. A completely new binding site for phosphate has also been identified in the complex with 2PG which differs substantially from the conventional phosphate binding site of the ligand in the crystal structures of TIM determined so far.     

Engineering of a single conserved amino acid residue of herpes simplex virus type 1 thymidine kinase allows a predominant shift from pyrimidine to purine nucleoside phosphorylation

Studies of herpes simplex virus type 1 (HSV-1) thymidine (dThd) kinase (TK) crystal structures show that purine and pyrimidine bases occupy distinct positions in the active site but approximately the same geometric plane. The presence of a bulky side chain, such as tyrosine at position 167, would not be sterically favorable for pyrimidine or pyrimidine nucleoside analogue binding, whereas purine nucleoside analogues would be less affected because they are located further away from the phenylalanine side chain. Site-directed mutagenesis of the conserved Ala-167 and Ala-168 residues in HSV-1 TK resulted in a wide variety of differential affinities and catalytic activities in the presence of the natural substrate dThd and the purine nucleoside analogue drug ganciclovir (GCV), depending on the nature of the amino acid mutation. A168H- and A167F-mutated HSV-1 TK enzymes turned out to have a virtually complete knock-out of dThd kinase activity (at least approximately 4-5 orders of magnitude lower) presumably due to a steric clash between the mutated amino acid and the dThd ring. In contrast, a full preservation of the GCV (and other purine nucleoside analogues) kinase activity was achieved for A168H TK. The enzyme mutants also markedly lost their binding capacity for dThd and showed a substantially diminished feedback inhibition by thymidine 5'-triphosphate. The side chain size at position 168 seems to play a less important role regarding GCV or dThd selectivity than at position 167. Instead, the nitrogen-containing side chains from A168H and A168K seem necessary for efficient ligand discrimination. This explains why A168H-mutated HSV-1 TK fully preserves its GCV kinase activity (Vmax/Km 4-fold higher than wild-type HSV-1 TK), although still showing a severely compromised dThd kinase activity (Vmax/Km 3-4 orders of magnitude lower than wild-type HSV-1 TK).     

Structural determinants for ligand binding and catalysis of triosephosphate isomerase.

The crystal structure of leishmania triosephosphate isomerase (TIM) complexed with 2-(N-formyl-N-hydroxy)-aminoethyl phosphonate (IPP) highlights the importance of Asn11 for binding and catalysis. IPP is an analogue of the substrate D-glyceraldehyde-3-phosphate, and it is observed to bind with its aldehyde oxygen in an oxyanion hole formed by ND2 of Asn11 and NE2 of His95. Comparison of the mode of binding of IPP and the transition state analogue phosphoglycolohydroxamate (PGH) suggests that the Glu167 side chain, as well as the triose part of the substrate, adopt different conformations as the catalysed reaction proceeds. Comparison of the TIM-IPP and the TIM-PGH structures with other liganded and unliganded structures also highlights the conformational flexibility of the ligand and the active site, as well as the conserved mode of ligand binding.     

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.     

Functional and structural changes due to a serine to alanine mutation in the active-site flap of enolase

Crystallographic and kinetic methods have been used to characterize a site-specific variant of yeast enolase in which Ser 39 in the active-site flap has been changed to Ala. In the wild-type enzyme, the carbonyl and hydroxyl groups of Ser 39 chelate the second equivalent of divalent metal ion, effectively anchoring the flap over the fully liganded active site. With Mg(2+) as the activating cation, S39A enolase has <0.01% of wild-type activity as reported previously [J.M. Brewer, C.V. Glover, M.J. Holland, L. Lebioda, Biochim. Biophys. Acta 1383 (2) (1998) 351-355]. Measurements of (2)H kinetic isotope effects indicate that the proton abstraction from 2-phosphoglycerate (2-PGA) is significantly rate determining. Analysis of the isotope effects provides information on the relative rates of formation and breakdown of the enolate intermediate. Moreover, assays with different species of divalent metal ions reveal that with S39A enolase (unlike the case of wild-type enolase), more electrophilic metal ions promote higher activities. The kinetic results with the S39A variant support the notions that a rate-limiting product release lowers the activity of wild-type enolase with more electrophilic metal ions and that the metal ions are used to acidify the C2-proton of 2-PGA. The S39A enolase was co-crystallized with Mg(2+) and the inhibitor phosphonoacetohydroxamate (PhAH). The structure was solved and refined at a resolution of 2.1 A. The structure confirms the conjecture that the active-site flap is opened in the mutant protein. PhAH chelates to both Mg ions as in the corresponding structure of the wild-type complex. Positions of the side chains of catalytic groups, Lys 345 and Glu 211, and of "auxiliary" residues Glu 168 and Lys 396 are virtually unchanged relative to the complex with the wild-type protein. His 159, which hydrogen bonds to the phosphonate oxygens in the wild-type complex, is 5.7 A from the closest phosphonate oxygen, and the loop (154-166) containing His 159 is shifted away from the active center. A peripheral loop, Glu 251-Gly 275, also moves to open access to the active site.     

The catalytic and conformational cycle of Aquifex aeolicus KDO8P synthase: role of the L7 loop

KDO8P synthase catalyzes the condensation of arabinose 5-phosphate (A5P) and phosphoenolpyruvate (PEP) to form the 8-carbon sugar KDO8P and inorganic phosphate (P(i)). The X-ray structure of the wild-type enzyme shows that when both PEP and A5P bind, the active site becomes isolated from the environment due to a conformational change of the L7 loop. The structures of the R106G mutant, without substrates, and with PEP and PEP plus A5P bound, were determined and reveal that in R106G closure of the L7 loop is impaired. The structural perturbations originating from the loss of the Arg(106) side chain point to a role of the L2 loop in stabilizing the closed conformation of the L7 loop. Despite the increased exposure of the R106G active site, no abnormal reaction of PEP with water was observed, ruling out the hypothesis that the primary function of the L7 loop is to shield the active site from bulk solvent during the condensation reaction. However, the R106G enzyme displays several kinetic abnormalities on both the substrate side (smaller K(m)(PEP), larger K(i)(A5P) and K(m)(A5P)) and the product side (smaller K(i)(Pi) and K(i)(KDO8P)) of the reaction. As a consequence, the mutant enzyme is less severely inhibited by A5P and more severely inhibited by P(i) and KDO8P. Simulations of the flux of KDO8P synthesis under metabolic steady-state conditions (constant concentration of reactants and products over time) suggest that in vivo R106G is expected to perform optimally in a narrower range of substrate and product concentrations than the wild-type enzyme.