Glu Side Chains Research Articles

Copper(II), nickel(II) and zinc(II) complexes of the N-terminal nonapeptide fragment of amyloid-β and its derivatives

Copper(II), nickel(II) and zinc(II) complexes of the nonapeptide fragment of amyloid-β Aβ(1–9) (NH2-DAEFRHDSG-NH2) and its two derivatives: NH2-DAAAAHAAA-NH2 and NH2-DAAAAAHAA-NH2 have been studied by potentiometric, UV–visible and CD spectroscopic methods. The results reveal the primary role of the amino terminus of peptides in copper(II) and nickel(II) binding. The formation of dinuclear complexes was also possible in the copper(II) containing systems but only the first six amino acids from the amino terminus were involved in metal binding in the physiologically relevant pH range. The coordination chemistry of the two alanine mutated peptides is almost the same as that of the native nonapeptide, but the thermodynamic stability of the copper(II) complexes of the mutants is significantly reduced. This difference probably comes from the secondary interactions of the polar side chains of Asp, Glu, Ser and Arg residues present in the native peptide. Moreover, this difference reveals that the amino acid sequence of the N-terminal domains of amyloid peptides is especially well suited for the complexation with copper(II) ions.

Arginyltransferase ATE1 Catalyzes Midchain Arginylation of Proteins at Side Chain Carboxylates In Vivo

Arginylation is an emerging posttranslational modification mediated by Arg-tRNA-protein-transferase (ATE1). It is believed that ATE1 links Arg solely to the N terminus of proteins, requiring prior proteolysis or action by Met-aminopeptidases to expose the arginylated site. Here, we tested the possibility of Arg linkage to midchain sites within intact protein targets and found that many proteins invivo are modified on the side chains of Asp and Glu by unconventional chemistry that targets the carboxy rather than the amino groups at the target sites. Such arginylation appears to be functionally regulated, and it can be directly mediated by ATE1, in addition to the more conventional ATE1-mediated linkage of Arg to the N-terminal alpha amino group. This midchain arginylation implies an unconventional mechanism of ATE1 action that likely facilitates its major biological role.

Strategic targeting of multiple water-mediated interactions: a concise and rational structure-based design approach to potent and selective MMP-13 inhibitors.

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Substrate Selectivity of the Acid-activated Glutamate/γ-Aminobutyric acid (GABA) Antiporter GadC from Escherichia coli

GadC, a central component of the Escherichia coli acid resistance system, is a Glu/GABA antiporter. A previous structural study and biochemical characterization showed that GadC exhibits a stringent pH dependence for substrate transport, with no detectable activity at pH values above 6.5. However, the substrate selectivity and the mechanism of pH-dependent transport activity of GadC remain enigmatic. In this study, we demonstrate that GadC selectively transports Glu with no net charge and GABA with a positive charge. A C-plug-truncated variant of GadC (residues 1-470) transported Gln (a mimic of Glu with no net charge), but not Glu, even at pH 8.0. The pH-dependent transport of Gln by this GadC variant was shifted ~1 unit toward a higher pH compared with Glu transport. Taken together, the results identify the substrate selectivity for GadC and show that the protonation states of substrates are crucial for transport.

Amino acid side chain induced selectivity in the hydrolysis of peptides catalyzed by a Zr(iv)-substituted Wells–Dawson type polyoxometalate

In this paper the reactivity of K15H[Zr(α2-P2W17O61)2]·25H2O (1), a Zr(IV)-substituted Wells-Dawson polyoxometalate, is examined towards a series of Gly-Aa, Aa-Gly or Aa-Ser dipeptides, in which the nature and the size of the Aa amino acid side chain were varied. The rate of peptide bond hydrolysis, determined by (1)H NMR experiments, in Gly-Aa dipeptides is strongly dependent on the molecular volume and the chemical structure of the Aa side chain. When the volume of the aliphatic side chain of the Aa residue in Gly-Aa increased, a clear decrease in the hydrolysis rate was observed. Replacing one α-H in the C-terminal Gly residue of Gly-Gly by a methyl group (Gly-Ala) resulted in a 6-fold reactivity decrease, pointing towards the importance of steric factors for efficient peptide bond hydrolysis. The rate constants for peptide bond hydrolysis in Gly-Aa dipeptides at pD 5.0 and 60 °C ranged from 208.0 ± 15.6 × 10(-6) min(-1) for Gly-Ser to 5.0 ± 1.0 × 10(-6) min(-1) for Gly-Glu, reflecting the influence of the different nature of the amino acid side chains on the hydrolysis rate. Faster hydrolysis was observed for peptides containing Ser and Thr since the hydroxyl group in their side chain is able to facilitate amide bond hydrolysis by promoting an N→O acyl rearrangement. Peptides containing positively charged side chains at pD 5.0 show enhanced hydrolysis rates as a result of the secondary electrostatic interactions with the negatively charged surface of the polyoxometalate, which stabilize the peptide-polyoxometalate complex. A slow hydrolysis rate was observed for Gly-Glu, because of the preferential coordination of the carboxylate group in the side chain of Glu to Zr(IV), which prevents coordination of the peptide carbonyl group and its activation towards hydrolysis.

Retention of Conformational Entropy upon Calmodulin Binding to Target Peptides Is Driven by Transient Salt Bridges

Calmodulin (CaM) is a highly flexible calcium-binding protein that mediates signal transduction through an ability to differentially bind to highly variable binding sequences in target proteins. To identify how binding affects CaM motions, and its relationship to conformational entropy and target peptide sequence, we have employed fully atomistic, explicit solvent molecular dynamics simulations of unbound CaM and CaM bound to five different target peptides. The calculated CaM conformational binding entropies correlate with experimentally derived conformational entropies with a correlation coefficient R2 of 0.95. Selected side-chain interactions with target peptides restrain interhelical loop motions, acting to tune the conformational entropy of the bound complex via widely distributed CaM motions. In the complex with the most conformational entropy retention (CaM in complex with the neuronal nitric oxide synthase binding sequence), Lys-148 at the C-terminus of CaM forms transient salt bridges alternating between Glu side chains in the N-domain, the central linker, and the binding target. Additional analyses of CaM structures, fluctuations, and CaM-target interactions illuminate the interplay between electrostatic, side chain, and backbone properties in the ability of CaM to recognize and discriminate against targets by tuning its conformational entropy, and suggest a need to consider conformational dynamics in optimizing binding affinities.

Elucidating the Locking Mechanism of Peptides onto Growing Amyloid Fibrils through Transition Path Sampling

We investigate the molecular mechanism of monomer addition to a growing amyloid fibril composed of the main amyloidogenic region from the insulin peptide hormone, the LVEALYL heptapeptide. Applying transition path sampling in combination with reaction coordinate analysis reveals that the transition from a docked peptide to a locked, fully incorporated peptide can occur in two ways. Both routes involve the formation of backbone hydrogen bonds between the three central amino acids of the attaching peptide and the fibril, as well as a reorientation of the central Glu side chain of the locking peptide toward the interface between two β-sheets forming the fibril. The mechanisms differ in the sequence of events. We also conclude that proper docking is important for correct alignment of the peptide with the fibril, as alternative pathways result in misfolding.

Effect of Glutamate Side Chain Length on Intrahelical Glutamate–Lysine Ion Pairing Interactions

Ion pairing interactions between oppositely charged amino acids are important for protein structure stability. Despite the apparent electrostatic nature of these interactions, the charged amino acids Lys, Arg, Glu, and Asp have a different number of hydrophobic methylenes linking the charged functionality to the backbone. To investigate the effect of Glu (and Asp) side chain length on ion pairing interactions, a series of 36 monomeric α-helical peptides containing Zbb-Xaa (i, i+3), (i, i+4), and (i, i+5) (Zbb = Aad, Glu, Asp; Xaa = Lys, Orn, Dab, Dap) sequence patterns were studied by circular dichroism (CD) spectroscopy at pH 7 and 2. Peptides with Glu and Aad exhibited similar helicity and pH dependence, whereas peptides with Asp behaved distinctly different. The side chain interaction energetics were derived from the CD data using the nesting block method coupled with modified Lifson-Roig theory. At pH 7, no Zbb-Xaa (i, i+5) interaction was observed, regardless of side chain length (consistent with the helix geometry). Interestingly, only Lys was capable of supporting Zbb-Xaa (i, i+3) interactions, whereas any Xaa side chain length supported Zbb-Xaa (i, i+4) interactions. In particular, the magnitude of both Zbb(-)-Lys (i, i+4) and Zbb(-)-Orn (i, i+4) interaction energies followed the trend Asp > Glu > Aad. Side chain conformational analysis by molecular mechanics calculations showed that the Zbb-Xaa (i, i+3) interactions involved the χ(1) dihedral combination (g+, g+) for the i and i+3 residues, whereas the Zbb-Xaa (i, i+4) interactions were supported by the χ(1) dihedral combination (t, g+) for the i and i+4 residues. These calculated low energy conformers were consistent with conformations of intrahelical Asp-Lys and Glu-Lys salt bridges in a nonredundant protein structure database. These results suggest that Asp and Glu provide natural variation, and lengthening the Glu side chain further to Aad does not furnish additional characteristics that Glu cannot supply.

X-ray Structures of Magnesium and Manganese Complexes with the N-Terminal Domain of Calmodulin: Insights into the Mechanism and Specificity of Metal Ion Binding to an EF-Hand

Calmodulin (CaM), a member of the EF-hand superfamily, regulates many aspects of cell function by responding specifically to micromolar concentrations of Ca(2+) in the presence of an ~1000-fold higher concentration of cellular Mg(2+). To explain the structural basis of metal ion binding specificity, we have determined the X-ray structures of the N-terminal domain of calmodulin (N-CaM) in complexes with Mg(2+), Mn(2+), and Zn(2+). In contrast to Ca(2+), which induces domain opening in CaM, octahedrally coordinated Mg(2+) and Mn(2+) stabilize the closed-domain, apo-like conformation, while tetrahedrally coordinated Zn(2+) ions bind at the protein surface and do not compete with Ca(2+). The relative positions of bound Mg(2+) and Mn(2+) within the EF-hand loops are similar to those of Ca(2+); however, the Glu side chain at position 12 of the loop, whose bidentate interaction with Ca(2+) is critical for domain opening, does not bind directly to either Mn(2+) or Mg(2+), and the vacant ligand position is occupied by a water molecule. We conclude that this critical interaction is prevented by specific stereochemical constraints imposed on the ligands by the EF-hand β-scaffold. The structures suggest that Mg(2+) contributes to the switching off of calmodulin activity and possibly other EF-hand proteins at the resting levels of Ca(2+). The Mg(2+)-bound N-CaM structure also provides a unique view of a transiently bound hydrated metal ion and suggests a role for the hydration water in the metal-induced conformational change.

Proton transfer reactions in carbon nanotubes endohedrally functionalized with selected polar amino acid sidechains

We use the empirical-valence-bond (EVB) theory to investigate intramolecular proton transfer reactions between a selected set of polar amino acid sidechains and hydroxyl groups suspended inside carbon nanotubes to model the effect of hydrophobic confinement on the energetics of proton transfer involving (i) translocation of an excess protonic charge (with protonated histidine sidechain as donor) and (ii) transformation of a neutral reactant state to a charge-separated product state (with sidechains of Asp, Glu, Ser and Thr as donor). In both the cases, confinement in hydrophobic medium is found to change the associated free energies compared to their respective values in the bulk solution phase. Presence of stable hydrogen bonding within the pore is found to have a significant effect on both free energies of reaction and activation and thus governs the thermodynamic and kinetic feasibilities of these intramolecular reactions in hydrophobic confinement.

Copper coordination to the putative cell binding site of angiogenin: a DFT investigation

We present a DFT study of the structural and spectroscopic properties of the complex formed by Cu2+ with the peptide fragment Ac-PHREN-NH2, which encompasses the putative cell binding domain of angiogenin, as well as with its Ac-PHRQN-NH2 variant. Analysis of structures, energies and spectroscopic parameters has allowed to conclude that the metal coordination environment at pH 8 is formed by a nitrogen atom of His, two deprotonated amide groups, and an oxygen atom from the COO− side chain of Glu, in nice agreement with recent experimental results (La Mendola et al. in Dalton Trans, 39:10678, 2010). Moreover, DFT results allowed to reveal that the Glu side chain of the Ac-PHREN-NH2 peptide is coordinated in equatorial position, in a tetrahedrically distorted square planar arrangement, fully disclosing the effects of Cu2+ binding on the structural properties of this key angiogenin portion. In the Ac-PHRQN-NH2 variant, the carboxylate group is replaced by a H2O molecule in a coordination arrangement similar to that of the wild-type system.

Cytosolic Carboxypeptidase 1 Is Involved in Processing α- and β-Tubulin

The Purkinje cell degeneration (pcd) mouse has a disruption in the gene encoding cytosolic carboxypeptidase 1 (CCP1). This study tested two proposed functions of CCP1: degradation of intracellular peptides and processing of tubulin. Overexpression (2-3-fold) or knockdown (80-90%) of CCP1 in human embryonic kidney 293T cells (HEK293T) did not affect the levels of most intracellular peptides but altered the levels of α-tubulin lacking two C-terminal amino acids (delta2-tubulin) ≥ 5-fold, suggesting that tubulin processing is the primary function of CCP1, not peptide degradation. Purified CCP1 produced delta2-tubulin from purified porcine brain α-tubulin or polymerized HEK293T microtubules. In addition, CCP1 removed Glu residues from the polyglutamyl side chains of porcine brain α- and β-tubulin and also generated a form of α-tubulin with two C-terminal Glu residues removed (delta3-tubulin). Consistent with this, pcd mouse brain showed hyperglutamylation of both α- and β-tubulin. The hyperglutamylation of α- and β-tubulin and subsequent death of Purkinje cells in pcd mice was counteracted by the knock-out of the gene encoding tubulin tyrosine ligase-like-1, indicating that this enzyme hyperglutamylates α- and β-tubulin. Taken together, these results demonstrate a role for CCP1 in the processing of Glu residues from β- as well as α-tubulin in vitro and in vivo.

Structural Basis for the Mg2+-Dependent Modulation of Calmodulin Activity

The physiological function of calmodulin (CaM) depends on its ability to respond specifically to micromolar Ca2+ signals in the presence of ∼1000-fold excess of the chemically similar Mg2+. The intracellular concentration of Mg2+ is tightly controlled in a narrow range of 0.5-1.0 mM, which is sufficient to fully or partially saturate the Ca2+-binding sites of many EF-hand proteins, including CaM, at the resting Ca2+ levels. To explain the mechanism of metal ion binding specificity we have compared the effects of Mg2+ (ionic radius r=0.72 A), Mn2+ (r=0.67 A) and Ca2+ (r=1.06 A) on the structure and stability of the N-terminal domain of calmodulin (N-CaM). The far UV-CD melting profiles show that all three metal ions stabilize the structure of N-CaM, as indicated by the concentration dependent increase in unfolding temperature from 52 °C (apo) to >100 °C, 95 °C and 75 °C in the presence of Ca2+, Mn2+ and Mg2+, respectively. We have determined the X-ray structures of N-CaM complexed with the three metal ions. Only the Ca2+-N-CaM complex features the open-domain active conformation, whereas Mg2+-N-CaM and Mn2+-N-CaM have a closed-domain, apo-like conformation. The relative positions of all bound metal ions with respect to the EF-hand-β-scaffold are similar, however the bidentate ligand, the Glu sidechain in the 12th position of the loop, interacts with Ca2+ only and does not bind directly to either Mn2+ or Mg2+. These results strongly support our proposal (Grabarek, BBA, 1813, 913, 2011) that the stereochemical constraints imposed by the two-EF-hand domain structure preclude Mg2+ from engaging the EF-hand ligands in the same way as Ca2+. Mg2+ stabilizes the apo-like conformation of an EF-hand, thus contributing to the switching off CaM activity at the resting Ca2+ concentrations.

Calmodulin Conformational Binding Entropy Is Driven by Transient Salt Bridges

Calmodulin (CaM) is a calcium-binding protein that mediates signal transduction through an ability to differentially bind to highly variable binding sequences in target proteins. Experimental measurements show a linear correlation between conformational entropy and overall binding entropy, suggesting that a predictive ability to calculate conformational entropy is critical to the identification of binding partners and their relative affinities. To identify how binding affects low- and high-frequency motions, and their relationships to conformational entropy, we have employed fully atomistic molecular dynamics simulations with explicit solvent. The calculated quasiharmonic entropies of CaM bound to the targets relative to unbound CaM are in agreement with experimentally derived conformational entropies, where extrapolation to infinite simulation time showed that at least 92% of the backbone entropy is captured over the course of the 100 ns simulation. Enhanced side chain entropy results from conformational disruption of the protein backbone within loop regions between sub-domain elements, acting to facilitate the formation of transient salt bridges between Lys148 at the C-terminus and GLU sidechains both in CaM helix A and GLU side-chains present in selected binding sequences (i.e., endothelial and neuronal nitric oxide synthases). Comparison of Cα root-mean-squared fluctuations and MET sidechain dihedral distributions of CaM bound to each of the targets revealed that MET sidechains have more torsional freedom when the backbone is more flexible, showing that MET sidechain rotation mirrors the overall conformational binding entropy. Taken together, these results help to illuminate the interplay between electrostatic, hydrophobic, backbone and sidechain properties in the ability of CaM to recognize and discriminate against targets by tuning its conformational entropy.

Contributions of Conserved Negative Surface Potentials in S2 and S3 to Voltage-Gating of Sodium Channels

The question of how S4 charges are stabilized in the hydrophobic membrane environment is of critical importance to understand the function of voltage-gated ion channels (VGICs). Besides lipids, proteinaceous interactions are thought to play an important role and, indeed, the electronegative surface potentials of aromatic side chains and the net negative charge of acidic side chains are well suited to fulfill this role. Interestingly, a single aromatic side chain in S2 and a number of acidic side chains in S2 and S3 are highly conserved among VGICs. These residues have been extensively studied in potassium channels but little is known about their role in sodium channels. Here we incorporate unnatural amino acids to probe their contributions to channel gating in the skeletal sodium channel isoform (Nav1.4). Replacing the S2 aromatic in domains I-IV with fluorinated Phe derivatives does not affect channel gating, ruling out a cation-pi interaction with S4 charges. Trp substitutions in this position lead to a significant right-shift in the GV in domain I only, highlighting not only the functional differences between Nav domains, but also sodium and potassium channels, as the latter show a dramatic left-shift in the GV when Trp is introduced at the equivalent position in S2. Furthermore, we replaced Glu side chains with the neutral isosteric Glu analog nitrohomolalanine (Nha). Our data show that neutralizing a negative charge in S2 near the extracellular end of S4 leads to a significant right-shift in the GV in domain I, while neutralizing negative charges in S2 and S3 near the intracellular end of S4 result in small or no shifts in the GV. Together, these results shed new light on our understanding of voltage-gating in sodium channels.

Can collision‐induced negative‐ion fragmentations of [M–H]– anions be used to identify phosphorylation sites in peptides?

A joint experimental and theoretical investigation of the fragmentation behaviour of energised [M-H](-) anions from selected phosphorylated peptides has confirmed some of the most complex rearrangement processes yet to be reported for peptide negative ions. In particular: pSer and pThr (like pTyr) may transfer phosphate groups to C-terminal carboxyl anions and to the carboxyl anion side chains of Asp and Glu, and characteristic nucleophilic/cleavage reactions accompany or follow these rearrangements. pTyr may transfer phosphate to the side chains of Ser and Thr. The reverse reaction, namely transfer of a phosphate group from pSer or pThr to Tyr, is energetically unfavourable in comparison. pSer can transfer phosphate to a non-phosphorylated Ser. The non-rearranged [M-H](-) species yields more abundant product anions than its rearranged counterpart. If a peptide containing any or all of Ser, Thr and Tyr is not completely phosphorylated, negative-ion cleavages can determine the number of phosphated residues, and normally the positions of Ser, Thr and Tyr, but not which specific residues are phosphorylated. This is in accord with comments made earlier by Lehmann and coworkers.

Low temperature FTIR spectroscopy provides new insights in the pH-dependent proton pathway of proteorhodopsin

In the presented study the low pH photocycle of proteorhodopsin is extensively investigated by means of low temperature FTIR spectroscopy. Besides the already well-known characteristics of the all-trans and 13-cis retinal vibrations the 77K difference spectrum at pH 5.1 shows an additional negative signal at 1744cm−1 which is interpreted as indicator for the L state. The subsequent photocycle steps are investigated at temperatures higher than 200K. The combination of visible and FTIR spectroscopy enabled us to observe that the deprotonation of the Schiff base is linked to the protonation of an Asp or Glu side chain — the new proton acceptor under acidic conditions. The difference spectra of the late intermediates are characterized by large amide I changes and two further bands ((−)1751cm−1/(+)1725cm-1) in the spectral region of the Asp/Glu ν(C=O) vibrations. The band position of the negative signature points to a transient deprotonation of Asp-97. In addition, the pH dependence of the acidic photocycle was investigated. The difference spectra at pH 5.5 show distinct differences connected to changes in the protonation state of key residues. Based on our data we propose a three-state model that explains the complex pH dependence of PR.

Molecular-level examination of Cu2+ binding structure for amyloid fibrils of 40-residue Alzheimer's β by solid-state NMR spectroscopy.

Cu(2+) binding to Alzheimer's β (Aβ) peptides in amyloid fibrils has attracted broad attention, as it was shown that Cu ion concentration elevates in Alzheimer's senile plaque and such association of Aβ with Cu(2+) triggers the production of neurotoxic reactive oxygen species (ROS) such as H(2)O(2). However, detailed binding sites and binding structures of Cu(2+) to Aβ are still largely unknown for Aβ fibrils or other aggregates of Aβ. In this work, we examined molecular details of Cu(2+) binding to amyloid fibrils by detecting paramagnetic signal quenching in 1D and 2D high-resolution (13)C solid-state NMR (SSNMR) for full-length 40-residue Aβ(1-40). Selective quenching observed in (13)C SSNMR of Cu(2+)-bound Aβ(1-40) suggested that primary Cu(2+) binding sites in Aβ(1-40) fibrils include N(ε) in His-13 and His-14 and carboxyl groups in Val-40 as well as in Glu sidechains (Glu-3, Glu-11, and/or Glu-22). (13)C chemical shift analysis demonstrated no major structural changes upon Cu(2+) binding in the hydrophobic core regions (residues 18-25 and 30-36). Although the ROS production via oxidization of Met-35 in the presence of Cu(2+) has been long suspected, our SSNMR analysis of (13)C(ε)H(3)-S- in M35 showed little changes after Cu(2+) binding, excluding the possibility of Met-35 oxidization by Cu(2+) alone. Preliminary molecular dynamics (MD) simulations on Cu(2+)-Aβ complex in amyloid fibrils confirmed binding sites suggested by the SSNMR results and the stabilities of such bindings. The MD simulations also indicate the coexistence of a variety of Cu(2+)-binding modes unique in Aβ fibril, which are realized by both intra- and intermolecular contacts and highly concentrated coordination sites due to the in-register parallel β-sheet arrangements.

Scope and limitation of side‐chain assisted ligation

Side-chain assisted ligation is an auxiliary-mediated ligation strategy in which a thiol bearing cyclohexane or cyclopentane is attached to the side-chain of Asp, Glu, Ser or Thr to function in a similar manner to Cys in a native chemical ligation. Following the ligation step, the auxiliary is removed, without product isolation, under alkaline conditions.

X-ray Structure and Mutational Analysis of the Atrazine Chlorohydrolase TrzN

Atrazine chlorohydrolase, TrzN (triazine hydrolase or atrazine chlorohydrolase 2), initiates bacterial metabolism of the herbicide atrazine by hydrolytic displacement of a chlorine substituent from the s-triazine ring. The present study describes crystal structures and reactivity of wild-type and active site mutant TrzN enzymes. The homodimer native enzyme structure, solved to 1.40 Å resolution, is a (βα)(8) barrel, characteristic of members of the amidohydrolase superfamily. TrzN uniquely positions threonine 325 in place of a conserved aspartate that ligates the metal in most mononuclear amidohydrolases superfamily members. The threonine side chain oxygen atom is 3.3 Å from the zinc atom and 2.6 Å from the oxygen atom of zinc-coordinated water. Mutation of the threonine to a serine resulted in a 12-fold decrease in k(cat)/K(m), largely due to k(cat), whereas the T325D and T325E mutants had immeasurable activity. The structure and kinetics of TrzN are reminiscent of carbonic anhydrase, which uses a threonine to assist in positioning water for reaction with carbon dioxide. An isosteric substitution in the active site glutamate, E241Q, showed a large diminution in activity with ametryn, no detectable activity with atratone, and a 10-fold decrease with atrazine, when compared with wild-type TrzN. Activity with the E241Q mutant was nearly constant from pH 6.0 to 10.0, consistent with the loss of a proton-donating group. Structures for TrzN-E241Q were solved with bound ametryn and atratone to 1.93 and 1.64 Å resolution, respectively. Both structure and kinetic determinations suggest that the Glu(241) side chain provides a proton to N-1 of the s-triazine substrate to facilitate nucleophilic displacement at the adjacent C-2.