Active Center Residues Research Articles

Crystal structure of restriction endonuclease Kpn2I of CCGG-family

BackgroundRestriction endonucleases belong to prokaryotic restriction-modification systems, that protect host cells from invading DNA. Type II restriction endonucleases recognize short 4–8 bp sequences in the target DNA and cut both DNA strands producing double strand breaks. Type II restriction endonuclease Kpn2I cleaves 5′-T/CCGGA DNA sequence (“/” marks the cleavage position). Analysis of protein sequences suggested that Kpn2I belongs to the CCGG-family, which contains ten enzymes that recognize diverse nucleotides outside the conserved 5′-CCGG core and share similar motifs for the 5′-CCGG recognition and cleavage. MethodsWe solved a crystal structure of Kpn2I in a DNA-free form at 2.88 Å resolution. From the crystal structure we predicted active center and DNA recognition residues and tested them by mutational analysis. We estimated oligomeric state of Kpn2I by SEC-MALS and performed plasmid DNA cleavage assay to elucidate DNA cleavage mechanism. ResultsStructure comparison confirmed that Kpn2I shares a conserved active site and structural determinants for the 5′-CCGG tetranucleotide recognition with other restriction endonucleases of the CCGG-family. Guided by structural similarity between Kpn2I and the CCGG-family restriction endonucleases PfoI and AgeI, Kpn2I residues involved in the outer base pair recognition were proposed. ConclusionsKpn2I is an orthodox Type IIP restriction endonuclease, which acts as a dimer. Kpn2I shares structural similarity to the CCGG-family restriction endonucleases PfoI, AgeI and PspGI. General significanceThe Kpn2I structure concluded the studies of the CCGG-family, covering detailed structural and biochemical characterization of eleven restriction enzymes and their complexes with DNA.

Optimization of Cholinesterase-Based Catalytic Bioscavengers Against Organophosphorus Agents.

Organophosphorus agents (OPs) are irreversible inhibitors of acetylcholinesterase (AChE). OP poisoning causes major cholinergic syndrome. Current medical counter-measures mitigate the acute effects but have limited action against OP-induced brain damage. Bioscavengers are appealing alternative therapeutic approach because they neutralize OPs in bloodstream before they reach physiological targets. First generation bioscavengers are stoichiometric bioscavengers. However, stoichiometric neutralization requires administration of huge doses of enzyme. Second generation bioscavengers are catalytic bioscavengers capable of detoxifying OPs with a turnover. High bimolecular rate constants (kcat/Km > 106 M−1min−1) are required, so that low enzyme doses can be administered. Cholinesterases (ChE) are attractive candidates because OPs are hemi-substrates. Moderate OP hydrolase (OPase) activity has been observed for certain natural ChEs and for G117H-based human BChE mutants made by site-directed mutagenesis. However, before mutated ChEs can become operational catalytic bioscavengers their dephosphylation rate constant must be increased by several orders of magnitude. New strategies for converting ChEs into fast OPase are based either on combinational approaches or on computer redesign of enzyme. The keystone for rational conversion of ChEs into OPases is to understand the reaction mechanisms with OPs. In the present work we propose that efficient OP hydrolysis can be achieved by re-designing the configuration of enzyme active center residues and by creating specific routes for attack of water molecules and proton transfer. Four directions for nucleophilic attack of water on phosphorus atom were defined. Changes must lead to a novel enzyme, wherein OP hydrolysis wins over competing aging reactions. Kinetic, crystallographic, and computational data have been accumulated that describe mechanisms of reactions involving ChEs. From these studies, it appears that introducing new groups that create a stable H-bonded network susceptible to activate and orient water molecule, stabilize transition states (TS), and intermediates may determine whether dephosphylation is favored over aging. Mutations on key residues (L286, F329, F398) were considered. QM/MM calculations suggest that mutation L286H combined to other mutations favors water attack from apical position. However, the aging reaction is competing. Axial direction of water attack is not favorable to aging. QM/MM calculation shows that F329H+F398H-based multiple mutants display favorable energy barrier for fast reactivation without aging.

Effect of ligand binding on the dynamics of trypsin. Comparison of different approaches

The intramolecular signal transduction induced by the binding of ligands to trypsin was investigated by molecular dynamics simulations. Ligand binding changes the residue-residue interaction energies and suppresses the mobility of loops that are in direct contact with the ligand. The reduced mobility of these loops results in the altered flexibility of the nearby loops and thereby transmits the information from ligand binding site to the remote sites. The analysis of the flexibility of all residues confirmed the coupling between loops L1 (185–188) and L2 (221–224) and the residues in the active center. The significance of S1 pocket residues for the signal transduction from the active center to the substrate-binding site was confirmed by the dynamical network and covariance matrix analyses. Gaussian network model and principal component analysis demonstrated that the active center residues had zero amplitude in the slowest fluctuations acting as hinges or anchors. Overall, our results provide a new insight into protein–ligand interactions and show how the allosteric signaling may occur.

Design, Synthesis and Mimic Catalytic Activity of a Vanadium Haloperoxidase Conformation Model Molecule

Based on mimicing the N, O coordination environment of the active center of vanadium haloperoxidase (V-HPOs) and the hydrogen-bond interaction between the active center and amino acid residues or water molecules, we designed and synthesized two kinds of oxidovanadium complexes: (C3H5N2)2[(VO)2(μ2-C2O4)(C2O4)2(H2O)2] (1) and (VO2)2(μ2-C2O4)(C3H4N2)2 (2). We determined their structures by X-ray single crystal diffraction. The crystal structure analysis indicated that the coordination environments of those oxidovanadium complexes were similar to the active center of vanadium haloperoxidase, and there seemed to be a mimicing α-helix structure in the three-dimensional packing structure of the complexes. Additionally, by studying the bromination reaction activity we found that the oxidovanadium complexes had an upper activity during the mimic catalytic process.

Biochemical and Structural Properties of Mouse Kynurenine Aminotransferase III

Kynurenine aminotransferase III (KAT III) has been considered to be involved in the production of mammalian brain kynurenic acid (KYNA), which plays an important role in protecting neurons from overstimulation by excitatory neurotransmitters. The enzyme was identified based on its high sequence identity with mammalian KAT I, but its activity toward kynurenine and its structural characteristics have not been established. In this study, the biochemical and structural properties of mouse KAT III (mKAT III) were determined. Specifically, mKAT III cDNA was amplified from a mouse brain cDNA library, and its recombinant protein was expressed in an insect cell protein expression system. We established that mKAT III is able to efficiently catalyze the transamination of kynurenine to KYNA and has optimum activity at relatively basic conditions of around pH 9.0 and at relatively high temperatures of 50 to 60 degrees C. In addition, mKAT III is active toward a number of other amino acids. Its activity toward kynurenine is significantly decreased in the presence of methionine, histidine, glutamine, leucine, cysteine, and 3-hydroxykynurenine. Through macromolecular crystallography, we determined the mKAT III crystal structure and its structures in complex with kynurenine and glutamine. Structural analysis revealed the overall architecture of mKAT III and its cofactor binding site and active center residues. This is the first report concerning the biochemical characteristics and crystal structures of KAT III enzymes and provides a basis toward understanding the overall physiological role of mammalian KAT III in vivo and insight into regulating the levels of endogenous KYNA through modulation of the enzyme in the mouse brain.

Remarkable enhancement in PLD activity from Streptoverticillium cinnamoneum by substituting serine residue into the GG/GS motif

The gene that encodes phospholipase D (PLD) from Streptoverticillium cinnamoneum contains three consensus regions (Region I, II and IV as shown in Fig. 1A) that are conserved among the PLD superfamily. The glycine–glycine (GG) motif in Region I and the glycine–serine (GS) motif in Region IV are also conserved in the PLD superfamily. These (GG and GS) motifs are located 7 residues downstream from each HKD motif. In an investigation of fifteen GG/GS motif mutants, generated as fusion proteins with maltose-binding protein (MBP–PLDs), three highly active mutants were identified. Three of the mutants (G215S, G216S, and G216S–S489G) contained a serine residue in the GG motif, and exhibited approximately a 9–27-fold increased transphosphatidylation activity to DPPC compared with recombinant wild type MBP–PLD. When heat stability was compared between three mutants and the recombinant wild type, only G216S–S489G showed heat labile properties. It appears that the 489th serine residue in the GS motif also contributes to the thermal stability of the enzyme. In addition, the GG/GS motif was very close to the active center residue, including two HKD motifs, as shown by computer modeling. The findings suggest that the GG/GS motif of PLD is a key motif that affects catalytic function and enzymatic stability.

Mechanism of allosteric regulation of transglutaminase 2 by GTP

Allosteric regulation is a fundamental mechanism of biological control. Here, we investigated the allosteric mechanism by which GTP inhibits cross-linking activity of transglutaminase 2 (TG2), a multifunctional protein, with postulated roles in receptor signaling, extracellular matrix assembly, and apoptosis. Our findings indicate that at least two components are involved in functionally coupling the allosteric site and active center of TG2, namely (i) GTP binding to mask a conformationally destabilizing switch residue, Arg-579, and to facilitate interdomain interactions that promote adoption of a compact, catalytically inactive conformation and (ii) stabilization of the inactive conformation by an uncommon H bond between a cysteine (Cys-277, an active center residue) and a tyrosine (Tyr-516, a residue located on a loop of the beta-barrel 1 domain that harbors the GTP-binding site). Although not essential for GTP-mediated inhibition of cross-linking, this H bond enhances the rate of formation of the inactive conformer.

Multiple Modes of Active Center Communication in Thiamin Diphosphate-Dependent Enzymes

Detection of interaction between cofactors at the active centers of homodimeric and homotetrameric enzymes is usually elusive by steady-state kinetic approaches and requires protein variants where such interactions are diminished or exaggerated. In this Account, evidence for active-center interactions will be presented for the following thiamin diphosphate-dependent enzymes: yeast pyruvate decarboxylase, benzoylformate decarboxylase, and examples from the 2-oxoacid dehydrogenase multienzyme complex class. The dissymmetry of active sites is especially evident in the X-ray structures of these enzymes with substrate/substrate analogues bound. Perturbations that reveal active center communication include use of chromophoric substrates and substitutions of active center residues on putative pathways.

Probing the open state of cytochrome P450cam with ruthenium-linker substrates.

Cytochromes P450 play key roles in drug metabolism and disease by oxidizing a wide variety of natural and xenobiotic compounds. High-resolution crystal structures of P450cam bound to ruthenium sensitizer-linked substrates reveal an open conformation of the enzyme that allows substrates to access the active center via a 22-A deep channel. Interactions of alkyl and fluorinated biphenyl linkers with the channel demonstrate the importance of exploiting protein dynamics for specific inhibitor design. Large changes in peripheral enzyme structure (F and G helices) couple to conformational changes in active center residues (I helix) implicated in proton pumping and dioxygen activation. Common conformational states among P450cam and homologous enzymes indicate that static and dynamic variability in the F/G helix region allows the 54 human P450s to oxidize thousands of substrates.

Studies on the enzymatic and transcriptional activity of the dimerization cofactor for hepatocyte nuclear factor 1.

The relationship between the enzymatic and the transcriptional activity of the bifunctional protein pterin-4a-carbinolamine dehydratase/dimerization cofactor for hepatocyte nuclear factor 1 (DCoH) has been elucidated by site-directed mutagenesis. DCoH dimers harbor a binding site for hepatocyte nuclear factor 1 (HNF1), two active centers that bind pterins, and a saddle-shaped surface that resembles nucleic acid binding domains. Two domains of the protein have been selectively targeted to determine if a change in one activity affects the other. No strong correlation has been found, supporting the idea that carbinolamine dehydratase activity is not required for HNF1 binding in vitro or transcriptional coactivation in vivo. Double mutations in the active center, however, influence the in vivo transcriptional activity but not HNF1 binding. This finding suggests that some active center residues also are used during transcription, possibly for binding of another (macro)molecule. Several mutations in the saddle led to a surprising increase in transcription, therefore linking this domain to transcriptional regulation as well. The transcriptional function of DCoH therefore is composed of two parts, HNF1 binding and another contributing effect that involves the active site and, indirectly, the saddle.

HPr: a model protein.

Histidine-containing protein (HPr) is a central component of the bacterial phosphoenolpyruvate: sugar phosphotransferase system (PTS). This brief review covers recent structure-function studies on the active center of this protein: the role of the active center residues in phosphotransferase; the residues contributing to the phosphohydrolysis properties of HPr; and the contribution residues in HPr make to the pKa of the transiently phosphorylated active-site residue, His 15. As well, the potential for HPr to be used as a model protein for studying problems not directly associated with its function in the PTS is discussed.

Solid‐State nuclear magnetic resonance investigation of solvent dependence of tyrosyl ring motion in an enzyme

Tyrosyl ring motions in alpha-lytic protease were investigated by solid-state deuterium nuclear magnetic resonance (NMR) spectroscopy in lyophilized enzyme powder, in powder suspended in organic solvents, and in aqueous crystals. Ring flipping rates were determined by examining deuterium quadrupole echo line shapes. Of the four Tyr residues in the enzyme, one was flipping at the slow (< or =10(3) s(-1)) and one at the fast (> or =10(7) s(-1)) exchange limit of the line shape experiment in all the environments tested. Flipping rates of the remaining two Tyr residues depended markedly on the solvent, with the lowest flipping rates (< or =10(3) s(-1) for both residues) observed in the enzyme powder, whether dry or suspended in hydrophobic tert-butyl methyl ether. In hydrophilic dioxane and acetonitrile, the mobility of these residues increased to 10(4) and 10(5) s(-1). The latter rate rose further to 10(6) s(-1) in the hydrated hydrophilic solvents and to > or =10(7) s(-1) in aqueous crystals. The deuterium spectrum of native alpha-lytic protease was compared with that of the enzyme whose active center was covalently modified with an inhibitor, which binds next to Tyr-123, constraining its ring. This experiment revealed that water addition to acetonitrile specifically increased the flipping rate of this active center residue. Librational motions ("wobbling"), estimated by their effect on spin-lattice relaxation times, were slowest in the anhydrous solvents, intermediate in the hydrated solvents, and fastest in the aqueous crystals. Thus, alpha-lytic protease is more rigid in organic solvents than in water, as judged by mobility of its tyrosyl residues. Water stripping by hydrophilic solvents did not increase enzyme rigidity, nor were there clear correlations between mobility and either enzymatic activity or solvent dielectric constant.

Peptidyl sulfonium salts. A new class of protease inhibitors.

The possibility has been examined that peptidylmethyl sulfonium salts might affinity label proteases by an alkyl transfer from sulfur to an active center residue. The synthesis of a number of agents of this type is described as well as initial results of their effect on cysteinyl proteases, papain and cathepsin B. These are readily inactivated by reagents in which the peptidyl portion contains features that promote binding to the proteases such as a penultimate phenylalanine residue. Irreversible inactivation ensues by transfer of the peptidyl portion, not methyl groups. Peptidylmethyl sulfonium salts lose a proton to form an ylide structure which may be the prevalent form at physiological pH values. The ylide may also be the active affinity labeling form of the reagent since the rate of inactivation of cathepsin B increases with pH. In contrast, the action of another affinity labeling reagent for cathepsin B, benzyloxycarbonyl-Phe-AlaCHN2, a diazomethyl ketone, is relatively independent of pH.

The structure of manganese superoxide dismutase from Thermus thermophilus HB8 at 2.4-A resolution.

An atomic model of tetrameric manganese superoxide dismutase from Thermus thermophilus HB8 has been built into an electron density map at 2.4 A resolution, using chemical sequences of Mn dismutases from Thermus aquaticus and Bacillus stearothermophilus. The monomer fold is structurally very similar to the fold of iron dismutase and comprises two domains, each contributing two ligands to the metal. The Mn(III) ion is bound by protein ligands assigned as His 28, His 83, Asp 165, and His 169. Near neighbors in the metal-ligand environment include a series of hydrophobic residues, Phe 86, Trp 87, Trp 131, and Trp 167. The hydroxyl groups of two Tyr residues, at 36 and 182, are less than 7 A from the metal, as is His 32. Gln 150 forms a bridge between Tyr 36 and Trp 131. These ligands and nearby residues are strongly conserved in the known sequences of Mn dismutases. Only one of the two oxygens of Asp 165 has been assigned as a metal ligand, so that in the current model four protein atoms bind Mn(III). These ligand atoms form part of an approximate trigonal bipyramid in which water may occupy an axial position on the side opposite His 28. The conformation of the protein is unusual in the vicinity of the first ligand, His 28, as a consequence of the insertion of an extra residue in an alpha-helix. The distortion of the helix allows His 32 to stack against the ligand, His 169, and brings Tyr 36 close to the Mn ion. Across one of the dimer interfaces, the two Mn ions are separated by about 18 A, and active center residues from adjoining subunits interdigitate; Tyr 172 interacts with His 32 of the neighboring chain and Glu 168 with the backbone of 168 and with the ligand His 169 from the opposite subunit. Only one other dimer interface occurs in the tetramer; it involves residues 55-62 and sequences near 140 and 156. The center of the oligomeric molecule is filled with solvent.

The refined crystal structure of dimeric phospholipase A2 at 2.5 A. Access to a shielded catalytic center.

The 2.5-A crystal structure of the calcium-free form of the dimeric venom phospholipase A2 from the Western Diamondback rattlesnake Crotalus atrox, has been refined to an R-factor of 17.8% (I greater than 2 sigma) and acceptable stereochemistry. The molecule is a nearly perfect 2-fold symmetric dimer in which most of the catalytic residues of both subunits face an internal cavity. The restricted access to the putative catalytic sites is especially puzzling as the optimal substrates for this and most other phospholipase A2 are phospholipids condensed in micellar or lamellar aggregates. We point out that substrate access to the internal cavity may be aided by calcium binding which can alter the intersubunit contacts that shield the catalytic network. We also suggest that a system of hydrogen-bonded moieties exists on the surface of the dimer that links the amino terminus to the catalytic system, through an invariant Gln 4 side chain and the backbone of the active center residue, Tyr 73. This hydrogen-bonded network is on a highly accessible surface of the dimer and would appear to contribute to the enzyme's (as opposed to the proenzyme's) special capacity to attack aggregated rather than monomeric substrate.

Evolution in the structure and function of carboxyl proteases

A model for the structure and function of extracellular carboxyl (acid) proteases can be established from three amino acid sequences and four crystal structures of these enzymes. The carboxyl proteases from gastric and fungal origins are very homologous in both primary and tertiary structures. The molecules consist of about 320 residues organized with a secondary structure which is primarily comprised of beta-strands and very similar tertiary structures. An apparent binding cleft, which can accommodate a substrate with about eight amino acid residues, contains near its midpoint the active center residues Asp-215, Asp-32, and Ser-35. These three residues are hydrogen bonded to each other. An intracellular carboxyl protease, cathepsin D, is very homologous to the extracellular enzymes in N-terminal amino acid sequence and primary structure location of active center residues. The tertiary structure of cathepsin D is probably similar, as well. However, cathepsin D contains a unique hydrophobic "tail" made up of about 100 residues added on the C-terminal side. Cathepsin D precursor is over 100,000 daltons in molecular weights, as contrasted to the gastric carboxyl protease zymogens, which are about 40,000 daltons. Carboxyl proteases contain two lobes symmetrical in peptide chain conformations. Each of the lobes also consists of two homologous structural units. These structural characteristics suggest that the original gene was coded for only about eighty amino acid residues and that gene duplication and fusion has taken place twice to produce a single chain carboxyl protease with four basic structural units in two symmetrical lobes. The formation of the zymogens and the cathepsin D "tail" must have resulted from various gene fusions. Partial sequence comparisons also suggest that cathepsin D may be an evolutionary ancestral chain for gastric carboxyl proteases.

Role of nascent alpha-ketoaldehyde in substrate-dependent oxidative inactivation of aldolase.

In the presence of electron acceptors rabbit muscle aldolase catalyzes the oxidation of dihydroxyacetone phosphate and in the course of this activity the enzyme is progressively inactivated [Christen, P., Cogoli-Greuter, M., Healy, M. J., and Lubini, D. (1976) Eur. J. Biochem. 63,223- 2311. We propose that the agent responsible for inactivation is the highly electrophilic a-ketoaldehyde produced in the reaction. This ketoaldehyde is herein shown to react rapidly with arginine sidechains, forming stable adducts, and with an active center residue in aldolase, leading to inactivation of the enzyme. Substrate-dependent oxidative inactivation of aldolase is brought about by reaction in situ of hydroxypyruvaldehyde phosphate with the essential residue located in the active site. The role of nascent hydroxypyruvaldehyde phosphate in aldolase inactivation is also borne out by studies with the substrate analogue D-5-ketofructose 1,6-bisphosphate. Cleavage of this hexose bisphosphate by aldolase yields hydroxypyruvaldehyde phosphate in the absence of any oxidant and, as in the case of oxidative production of the ketoaldehyde, the catalytic cycles lead to inactivation of the enzyme. A similar role is suggested for nascent ketoaldehydes in the substrate-dependent oxidative inactivation of class I1 aldolase of yeast and transaldolase. Possible physiological significance of a-ketoaldehyde production is discussed. Carbanionic enzyme-substrate intermediates are susceptible to oxidation : several enzymes forming such intermediates were shown to catalyze the oxidation of their substrates in the presence of suitable electron acceptors [l]. The carbanion intermediate of aldolase-dihydroxyacetone phosphate complex is also oxidized by a number of redox indicators [1,2], dihydroxyacetone phosphate being converted to hydroxypyruvaldehyde phosphate. Concomitant to this unusual ‘dehydrogenase’ activity aldolase is inactivated [3]. The action of the inactivating agent was found to be practically restricted to the enzyme molecule that has produced it, suggesting that it is a transiently reactive entity. It was therefore concluded that an oxidatively activated intermediate, tentatively identified as a free radical, is responsible for the covalent modification and inactivation of aldolase [3]. This paper presents evidence for a role of hydroxypyruvaldehyde phosphate in the inactivation of aldolase. This ketoaldehyde reacts with arginine residues

The reaction of sulfhydryl reagents with bovine hepatic monoamine oxidase: Evidence for the presence of two cysteine residues essential for activity

The bovine liver monoamine oxidase (EC 1.4.3.4) was found to be inactivated by various well-known sulfhydryl reagents like p- mercuribenzoate , methylmercuric idodide and 5,5′-dithiobis-(2-nitro benzoic acid). The present investigation shows that the inactivation of the enzyme results from reactions of these reagents with 2 out of 8 titratable sulfhydryl groups per 10 5 g of the enzyme. The substrate, benzylamine, and competitive inhibitors like benzaldehyde, p- nitrobenzaldehyde , benzyl alcohol protected the enzyme from inactivation by the mercurials or the Ellman reagent. The inactivation experiments with these sulfhydryl reagents, the protection experiments, and the kinetics as well as physicochemical observations suggest that there are only two cysteine residues that are required for activity of the enzyme. It is possible that these two residues may be active-center residues.

The Primary Structure of Human Plasminogen: II. THE HISTIDINE LOOP OF HUMAN PLASMIN: LIGHT (B) CHAIN ACTIVE CENTER HISTIDINE SEQUENCE

Abstract Three peptide fragments were obtained from the S-carboxymethylated light (B) chain derivative of human plasmin after treatment with cyanogen bromide. The major cyanogen bromide fragment, containing over 80% of the amino acid residues, was found to contain the 3H-radiolabeled l-1-chloro-3-tosylamido-7-amino-2-heptanone active site. The active center residue was determined to be the NH2-terminal 18th residue of the major cyanogen bromide fragment. The NH2-terminal 20 residues of this fragment were determined to be -His-Phe-Cys-Gly-Gly-Thr-Leu-Ile-Ser-Pro-Glu-Trp-Val-Leu-Ser-Ala-Ala-His*-Cys-Leu-. This sequence contains the histidine of human plasmin; it is homologous to the loop sequence of other serine proteases.

Characterization of porcine malate dehydrogenase I. An active center peptide

Iodoacetamide has been found to be a highly useful reagent for the selective chemical modification of active center residues of porcine heart mitochondrial malate dehydrogenase ( l-malate:NAD + oxidoreductase, EC 1.1.1.37). The addition of the coenzyme, NADH, fully protects the enzyme from alkylation thus suggesting that chemical modification is occurring at or near the NADH binding site. Alkylation by this reagent leads to a totally inactive enzyme. The identification of histidine as the site of chemical modification confirms work recently reported by B.H. Anderton ( Eur. J. Biochem., 15 (1970) 562). However, in addition to the alkylated histidine, we have isolated a small active center peptide containing 2 residues of glycine in addition to carboxymethyl histidine. The composition and structure of this peptide was identified after difficulties arose in attempts to identify the active center residues derived from proteolytic digestion of the iodoacetamide modiffied enzyme.