Crystal structure of glycerophosphodiester phosphodiesterase (GDPD) from Thermoanaerobacter tengcongensis, a metal ion-dependent enzyme: insight into the catalytic mechanism

Glycerophosphodiester phosphodiesterase (GDPD; EC 3.1.4.46) catalyzes the hydrolysis of a glycerophosphodiester to an alcohol and glycerol 3-phosphate in glycerol metabolism. It has an important role in the synthesis of a variety of products that participate in many biochemical pathways. We report the crystal structure of the Thermoanaerobacter tengcongensis GDPD (ttGDPD) at 1.91 A resolution, with a calcium ion and glycerol as a substrate mimic coordinated at this calcium ion (PDB entry 2pz0). The ttGDPD dimer with an intermolecular disulfide bridge and two hydrogen bonds is considered as the potential functional unit. We used site-directed mutagenesis to characterize ttGDPD as a metal ion-dependent enzyme, identified a cluster of residues involved in substrate binding and the catalytic reaction, and we propose a possible general acid-base catalytic mechanism for ttGDPD. Superposing the active site with the homologous structure GDPD from Agrobacterium tumefaciens (PDB entry 1zcc), which binds a sulfate ion in the active site, the sulfate ion can represent the phosphate moiety of the substrate, simulating the binding mode of the true substrate of GDPD.     

Defining the catalytic metal ion interactions in the Tetrahymena ribozyme reaction

Divalent metal ions play a crucial role in catalysis by many RNA and protein enzymes that carry out phosphoryl transfer reactions, and defining their interactions with substrates is critical for understanding the mechanism of biological phosphoryl transfer. Although a vast amount of structural work has identified metal ions bound at the active site of many phosphoryl transfer enzymes, the number of functional metal ions and the full complement of their catalytic interactions remain to be defined for any RNA or protein enzyme. Previously, thiophilic metal ion rescue and quantitative functional analyses identified the interactions of three active site metal ions with the 3'- and 2'-substrate atoms of the Tetrahymena group I ribozyme. We have now extended these approaches to probe the metal ion interactions with the nonbridging pro-S(P) oxygen of the reactive phosphoryl group. The results of this study combined with previous mechanistic work provide evidence for a novel assembly of catalytic interactions involving three active site metal ions. One metal ion coordinates the 3'-departing oxygen of the oligonucleotide substrate and the pro-S(P) oxygen of the reactive phosphoryl group; another metal ion coordinates the attacking 3'-oxygen of the guanosine nucleophile; a third metal ion bridges the 2'-hydroxyl of guanosine and the pro-S(P) oxygen of the reactive phosphoryl group. These results for the first time define a complete set of catalytic metal ion/substrate interactions for an RNA or protein enzyme catalyzing phosphoryl transfer.     

Structural and catalytic diversity within the amidohydrolase superfamily

The amidohydrolase superfamily comprises a remarkable set of enzymes that catalyze the hydrolysis of a wide range of substrates bearing amide or ester functional groups at carbon and phosphorus centers. The most salient structural landmark for this family of hydrolytic enzymes is a mononuclear or binuclear metal center embedded within the confines of a (beta/alpha)(8)-barrel structural fold. Seven variations in the identity of the specific amino acids that function as the direct metal ligands have been structurally characterized by X-ray crystallography. The metal center in this enzyme superfamily has a dual functionality in the expression of the overall catalytic activity. The scissile bond of the substrate must be activated for bond cleavage, and the hydrolytic water molecule must be deprotonated for nucleophilic attack. In all cases, the nucleophilic water molecule is activated through complexation with a mononuclear or binuclear metal center. In the binuclear metal centers, the carbonyl and phosphoryl groups of the substrates are polarized through Lewis acid catalysis via complexation with the beta-metal ion, while the hydrolytic water molecule is activated for nucleophilic attack by interaction with the alpha-metal ion. In the mononuclear metal centers, the substrate is activated by proton transfer from the active site, and the water is activated by metal ligation and general base catalysis. The substrate diversity is dictated by the conformational restrictions imposed by the eight loops that extend from the ends of the eight beta-strands.     

The reaction pathway of the isomerization of d-xylose catalyzed by the enzyme d-xylose isomerase: A theoretical study

Different pathways of the metal-induced isomerization of D-xylose to D-xylulose are investigated and compared in detail using energy minimization and molecular dynamics simulation. Two theoretical models are constructed for the reaction: in vacuum and in the enzyme D-xylose isomerase. The vacuum model is constructed based on the X-ray structure of the active site of D-xylose isomerase. It contains the atoms directly involved in the reaction and is studied using a semi-empirical molecular orbital method (PM3). The model in the enzyme includes the effects of the enzyme environment on the reaction using a combined quantum mechanical and molecular mechanical potential. For both models, the structures of the reactants, products, and intermediate complexes along the isomerization pathway are optimized. The effects of the position of the “catalytic Mg²⁺ ion” on the energies of the reactions are studied. The results indicate: 1) in vacuum, the isomerization reaction is favored when the catalytic metal cation is at site A, which is remote from the substrate; 2) in the enzyme, the catalytic metal cation, starting from site A, moves and stays at site B, which is close to the substrate; analysis of the charge redistribution of the active site during the catalytic process shows that the metal ion acts as a Lewis acid to polarize the substrate and catalyze the hydride shift; these results are consistent with previous experimental observations; and 3) Lys183 plays an important role in the isomerization reaction. The ϵ-NH₃⁺ group of its side chain can provide a proton to the carboxide ion of the substrate to form a hydroxyl group after the hydride shift step. This role of Lys183 has not been suggested before. Based on our calculations, we believe that this is a reasonable mechanism and consistent with site-directed mutation experiments. © 1997 Wiley-Liss Inc.     

Studies of the mechanism of action and regulation of cAMP-dependent protein kinase

Magnetic resonance and kinetic studies of the catalytic subunit of a Type II cAMP-dependent protein kinase from bovine heart have established the active complex to be an enzyme-ATP-metal bridge. The metal ion is β,γ coordinated with Δ chirality at the β-phosphorous atom. The binding of a second metal ion at the active site which bridges the enzyme to the three phosphoryl groups of ATP, partially inhibits the reaction. Binding of the metal-ATP substrate to the enzyme occurs in a diffusion-controlled reaction followed by a 40 ° change in the glycosidic torsional angle. This conformational change results from strong interaction of the nucleotide base with the enzyme. NMR studies of four ATP-utilizing enzymes show a correlation between such conformational changes and high nucleotide base specificity. Heptapeptide substrates and substrate analogs bind to the active site of the catalytic subunit at a rate significantly lower than collision frequency indicating conformational selection by the enzyme or a subsequent slow conformational change. NMR studies of the conformation of the enzyme-bound peptide substrates have ruled out α-helical and β-pleated sheet structures. The results of kinetic studies of peptide substrates in which the amino acid sequence was systematically varied were used to rule out the obligatory requirement for all possible β-turn conformations within the heptapeptide although an enzymatic preference for a β_2–5 or β_3–6 turn could not be excluded. Hence if protein kinase has an absolute requirement for a specific secondary structure, then this structure must be a coil. In the enzyme-substrate complex the distance along the reaction coordinate between the γ-P of ATP and the serine oxygen of the peptide substrate (5.3 ± 0.7 Å) allows room for a metaphosphate intermediate. This finding together with kinetic observations as well as the location of the inhibitory metal suggest a dissociative mechanism for protein kinase, although a mechanism with some associative character remains possible. Regulation of protein kinase is accomplished by competition between the regulatory subunit and peptide or protein substrates at the active site of the catalytic subunit. Thus, the regulatory subunit is found by NMR to block the binding of the peptide substrate to the active site of protein kinase but allows the binding of the nucleotide substrate and divalent cations. The dissociation constant of the regulatory subunit from the active site (10^?10m) is increased ~10-fold by phosphorylation and ~10⁴-fold by the binding of cAMP, to a value (10^?5m) which exceeds the intracellular concentration of the R₂C₂ holoenzyme complex (10^?6m). The resulting dissociation of the holoenzyme releases the catalytic subunit, permitting the active site binding of peptide or protein substrates.     

Higher metal-ligand coordination in the catalytic site of cobalt-substituted Thermoanaerobacter brockii alcohol dehydrogenase lowers the barrier for enzyme catalysis

Thermoanaerobacter brockii alcohol dehydrogenase (TbADH) is a zinc-dependent NADP(+)/H-linked class enzyme that reversibly catalyzes the oxidation of secondary alcohols to their corresponding ketones. Cobalt substitution studies of other members of the alcohol dehydrogenase (ADH) family showed that the cobalt-containing ADHs have a similar active site structure but slightly decreased activity compared to wild-type zinc ADHs. In contrast, the cobalt-substituted TbADH (Co-TbADH) exhibits an increase in specific activity compared to the native enzyme [Bogin, O., Peretz, M., and Burstein, Y. (1997) Protein Sci. 6, 450-458]. However, the structural basis underlying this behavior is not yet clear. To shed more light on this issue, we studied the local structure and electronics at the catalytic metal site in Co-TbADH by combining X-ray absorption (XAS) and quantum chemical calculations. Importantly, we show that the first metal-ligand coordination shell of Co-TbADH is distorted compared to its native tetrahedral coordination shell and forms an octahedral structure. This is mediated presumably by the addition of two water molecules and results in more positively charged catalytic metal ions. Recently, we have shown that the metal-ligand coordination number of the zinc ion in TbADH changes dynamically during substrate turnover. These structural changes are associated with a higher coordination number of the native catalytic zinc ion and the consequent buildup of a positive charge. Here we propose that the accumulation of a higher coordination number and positive charge at the catalytic metal ion in TbADH stabilizes the structure of the catalytic transition state and hence lowers the barrier for enzyme catalysis.     

Structure-based analysis of the metal-dependent mechanism of H-N-H endonucleases

Controversy surrounds the metal-dependent mechanism of H-N-H endonucleases, enzymes involved in a variety of biological functions, including intron homing and DNA repair. To address this issue we determined the crystal structures for complexes of the H-N-H motif containing bacterial toxin colicin E9 with Zn(2+), Zn(2+).DNA, and Mg(2+).DNA. The structures show that the rigid V-shaped architecture of the active site does not undergo any major conformational changes on binding to the minor groove of DNA and that the same interactions are made to the nucleic acid regardless of which metal ion is bound to the enzyme. The scissile phosphate contacts the single metal ion of the motif through distortion of the DNA brought about by the insertion of the Arg-96-Glu-100 salt bridge into the minor groove and a network of contacts to the DNA phosphate backbone that straddle the metal site. The Mg(2+)-bound structure reveals an unusual coordination scheme involving two H-N-H histidine residues, His-102 and His-127. The mechanism of DNA cleavage is likely related to that of other single metal ion-dependent endonucleases, such as I-PpoI and Vvn, although in these enzymes the single alkaline earth metal ion is coordinated by oxygen-bearing amino acids. The structures also provide a rationale as to why H-N-H endonucleases are inactive in the presence of Zn(2+) but active with other transition metal ions such as Ni(2+). This is because of coordination of the Zn(2+) ion through a third histidine, His-131. "Active" transition metal ions are those that bind more weakly to the H-N-H motif because of the disengagement of His-131, which we suggest allows a water molecule to complete the catalytic cycle.     

Crystal structure and functional analysis of the SurE protein identify a novel phosphatase family

Homologs of the Escherichia coli surE gene are present in many eubacteria and archaea. Despite the evolutionary conservation, little information is available on the structure and function of their gene products. We have determined the crystal structure of the SurE protein from Thermotoga maritima. The structure reveals the dimeric arrangement of the subunits and an active site around a bound metal ion. We also demonstrate that the SurE protein exhibits a divalent metal ion-dependent phosphatase activity that is inhibited by vanadate or tungstate. In the vanadate- and tungstate-complexed structures, the inhibitors bind adjacent to the divalent metal ion. Our structural and functional analyses identify the SurE proteins as a novel family of metal ion-dependent phosphatases.     

Catalytic features of the botulinum neurotoxin A light chain revealed by high resolution structure of an inhibitory peptide complex

The Clostridium botulinum neurotoxin serotype A light chain (BoNT/A-LC) is a Zn(II)-dependent metalloprotease that blocks the release of acetylcholine at the neuromuscular junction by cleaving SNAP-25, one of the SNARE proteins required for exocytosis. Because of the potential for use of the toxin in bioterrorism and the increasingly widespread application of the toxin in the medical field, there is significant interest in the development of small-molecule inhibitors of the metalloprotease. Efforts to design such inhibitors have not benefited from knowledge of how peptides bind to the active site since the enzyme-peptide structures available previously either were not occupied in the vicinity of the catalytic Zn(II) ion or did not represent the product of SNAP-25 substrate cleavage. Herein we report the 1.4 A-resolution X-ray crystal structure of a complex between the BoNT/A-LC and the inhibitory peptide N-Ac-CRATKML, the first structure of the light chain with an inhibitory peptide bound at the catalytic Zn(II) ion. The peptide is bound with the Cys S gamma atom coordinating the metal ion. Surprisingly, the cysteine sulfur is oxidized to the sulfenic acid form. Given the unstable nature of this species in solution, is it likely that oxidation occurs on the enzyme. In addition to the peptide-bound structure, we report two structures of the unliganded light chain with and without the Zn(II) cofactor bound at 1.25 and 1.20 A resolution, respectively. The two structures are nearly identical, confirming that the Zn(II) ion plays a purely catalytic role. Additionally, the structure of the Zn(II)-bound uncomplexed enzyme allows identification of the catalytic water molecule and a second water molecule that occupies the same position as the peptidic oxygen in the tetrahedral intermediate. This observation suggests that the enzyme active site is prearranged to stabilize the tetrahedral intermediate of the protease reaction.     

The reaction mechanism of the Ga(III)Zn(II) derivative of uteroferrin and corresponding biomimetics

Purple acid phosphatase from pig uterine fluid (uteroferrin), a representative of the diverse family of binuclear metallohydrolases, requires a heterovalent Fe(III)Fe(II) center for catalytic activity. The active-site structure and reaction mechanism of this enzyme were probed with a combination of methods including metal ion replacement and biomimetic studies. Specifically, the asymmetric ligand 2-bis{[(2-pyridylmethyl)-aminomethyl]-6-[(2-hydroxybenzyl)(2-pyridylmethyl)]aminomethyl}-4-methylphenol and two symmetric analogues that contain the softer and harder sites of the asymmetric unit were employed to assess the site selectivity of the trivalent and divalent metal ions using (71)Ga NMR, mass spectrometry and X-ray crystallography. An exclusive preference of the harder site of the asymmetric ligand for the trivalent metal ion was observed. Comparison of the reactivities of the biomimetics with Ga(III)Zn(II) and Fe(III)Zn(II) centers indicates a higher turnover for the former, suggesting that the M(III)-bound hydroxide acts as the reaction-initiating nucleophile. Catalytically active Ga(III)Zn(II) and Fe(III)Zn(II) derivatives were also generated in the active site of uteroferrin. As in the case of the biomimetics, the Ga(III) derivative has increased reactivity, and a comparison of the pH dependence of the catalytic parameters of native uteroferrin and its metal ion derivatives supports a flexible mechanistic strategy whereby both the mu-(hydr)oxide and the terminal M(III)-bound hydroxide can act as nucleophiles, depending on the metal ion composition, the geometry of the second coordination sphere and the substrate.     

The pH-dependence of the Escherichia coli RNase HII-catalysed reaction suggests that an active site carboxylate group participates directly in catalysis

RNase HII specifically catalyses the hydrolysis of phosphate diester linkages contained within the RNA portion of DNA/RNA hybrids. The catalytic parameters of the enzyme derived from Escherichia coli BL21 have been measured using 5'-fluorescent oligodeoxynucleotide substrates containing embedded ribonucleotides. The products of the reaction and the chemistry of phosphate diester hydrolysis were assigned unequivocally using mass spectrometry. The pH-dependence of the catalytic parameters was measured under conditions of optimal magnesium ion concentration. The logarithm of the turnover number of the enzyme increases steeply with pH until a pH-independent region is reached close to neutrality. The slope of the pH-dependent region is 2, indicating that the catalytically proficient form of RNase HII is di-anionic. The pH-dependence of log 1/K(M) is a sigmoidal curve reaching a maximal value at higher pH, suggesting deprotonation of a residue stabilises substrate binding. Possible mechanisms for the RNase HII-catalysed reaction consistent with the pH-dependent behaviour of the enzyme are discussed. The active sites of RNase H enzymes contain a cluster of four strictly conserved carboxylate groups. Together, the data suggest a requirement for ionisation of an active site carboxylic acid for metal ion binding or correct positioning of metal ion(s) in the enzyme-substrate complex and a role for a second active site carboxylate in general base catalysis.     

Structure and function of malic enzymes, a new class of oxidative decarboxylases

Malic enzyme is a tetrameric protein with double dimer structure in which the dimer interface is more intimately contacted than the tetramer interface. Each monomeric unit of the enzyme is composed of four structural domains, which show a different folding topology from those of the other oxidative decarboxylases. The active center is located at the interface between domains B and C. For human mitochondrial malic enzyme, there is an exo nucleotide-binding site for the inhibitor ATP and an allosteric site for the activator fumarate, located at the tetramer and dimer interfaces, respectively. Crystal structures of the enzyme in various complexed forms indicate that the enzyme may exist in equilibrium among two open and two closed forms. Interconversion among these forms involves rigid-body movements of the four structural domains. Substrate binding at the active site shifts the open form to the closed form that represents an active site closure. Fumarate binding at the allosteric site induces the interconversion between forms I and II, which is mediated by the movements of domains A and D. Structures of malic enzyme from different sources are compared with an emphasis on the differences and their implications to structure-function relationships. The binding modes of the substrate, product, cofactors, and transition-state analogue at the active site, as well as ATP and fumarate at the exo site and allosteric site, respectively, provide a clear account for the catalytic mechanism, nucleotide specificities, allosteric regulation, and functional roles of the quaternary structure. The proposed catalytic mechanism involves tyrosine-112 and lysine-183 as the general acid and base, respectively. In addition, a divalent metal ion (Mn(2+) or Mg(2+)) is essential in helping the catalysis. Binding of the metal ion also plays an important role in stabilizing the quaternary structural integrity of the enzyme.     

Mechanistic inferences from the binding of ligands to LpxC, a metal-dependent deacetylase

The metal-dependent deacetylase LpxC catalyzes the first committed step of lipid A biosynthesis in Gram-negative bacteria. Accordingly, LpxC is an attractive target for the development of inhibitors that may serve as potential new antibiotics for the treatment of Gram-negative bacterial infections. Here, we report the 2.7 A resolution X-ray crystal structure of LpxC complexed with the substrate analogue inhibitor TU-514 and the 2.0 A resolution structure of LpxC complexed with imidazole. The X-ray crystal structure of LpxC complexed with TU-514 allows for a detailed examination of the coordination geometry of the catalytic zinc ion and other enzyme-inhibitor interactions in the active site. The hydroxamate group of TU-514 forms a bidentate chelate complex with the zinc ion and makes hydrogen bond interactions with conserved active site residues E78, H265, and T191. The inhibitor C-4 hydroxyl group makes direct hydrogen bond interactions with E197 and H58. Finally, the C-3 myristate moiety of the inhibitor binds in the hydrophobic tunnel of the active site. These intermolecular interactions provide a foundation for understanding structural aspects of enzyme-substrate and enzyme-inhibitor affinity. Comparison of the TU-514 complex with cacodylate and imidazole complexes suggests a possible substrate diphosphate binding site and highlights residues that may stabilize the tetrahedral intermediate and its flanking transition states in catalysis. Evidence of a catalytic zinc ion in the native zinc enzyme coordinated by H79, H238, D242, and two water molecules with square pyramidal geometry is also presented. These results suggest that the native state of this metallohydrolase may contain a pentacoordinate zinc ion, which contrasts with the native states of archetypical zinc hydrolases such as thermolysin and carboxypeptidase A.     

Physicochemical Characterization of a Thermostable Alcohol Dehydrogenase from Pyrobaculum aerophilum

In this work we characterize an alcohol dehydrogenase (ADH) from the hyperthermophilic archaeon Pyrobaculum aerophilum (PyAeADHII). We have previously found that PyAeADHII has no activity when standard ADH substrates are used but is active when α-tetralone is used as substrate. Here, to gain insights into enzyme function, we screened several chemical libraries for enzymatic modulators using an assay employing α-tetralone. The results indicate that PyAeADHII activity in the presence of α-tetralone was inhibited by compounds such as flunarizine. We also examined metal coordination of the enzyme in solution by performing metal substitution of the enzyme-bound zinc (Zn²⁺) with cobalt. The solution-based absorption spectra for cobalt substituted PyAeADHII supports substitution at the structural Zn²⁺ site. To gain structural insight, we obtained the crystal structure of both wild-type and cobalt-substituted PyAeADHII at 1.75 Å and 2.20 Å resolution, respectively. The X-ray data confirmed one metal ion per monomer present only at the structural site with otherwise close conservation to other ADH enzymes. We next determined the co-crystal structure of the NADPH-bound form of the enzyme at 2.35 Å resolution to help define the active site region of the enzyme and this data shows close structural conservation with horse ADH, despite the lack of a catalytic Zn²⁺ ion in PyAeADHII. Modeling of α-tetralone into the NADPH bound structure suggests an arginine as a possible catalytic residue. The data presented here can yield a better understanding of alcohol dehydrogenases lacking the catalytic zinc as well as the structural features inherent to thermostable enzymes.     

Molecular comparison of a nonhemolytic and a hemolytic phospholipase C from Pseudomonas aeruginosa.

Pseudomonas aeruginosa produces two secreted phospholipase C (PLC) enzymes. The expression of both PLCs is regulated by Pi. One of the PLCs is hemolytic, and one is nonhemolytic. Low-stringency hybridization studies suggested that the genes encoding these two PLCs shared DNA homology. This information was used to clone plcN, the gene encoding the 77-kilodalton nonhemolytic PLC, PLC-N. A fragment of plcN was used to mutate the chromosomal copy of plcN by the generation of a gene interruption mutation. This mutant produces 55% less total PLC activity than the wild type, confirming the successful cloning of plcN. plcN was sequenced and encodes a protein which is 40% identical to the hemolytic PLC (PLC-H). The majority of the homology lies within the NH2 two-thirds of the proteins, while the remaining third of the amino acid sequence of the two proteins shows very little homology. Both PLCs hydrolyze phosphatidylcholine; however, each enzyme has a distinct substrate specificity. PLC-H hydrolyzes sphingomyelin in addition to phosphatidylcholine, whereas PLC-N is active on phosphatidylserine as well as phosphatidylcholine. These studies suggest structure-function relationships between PLC activity and hemolysis.     

Crystal structures of phosphate, iodide and iodate-inhibited phospholipase C from Bacillus cereus and structural investigations of the binding of reaction products and a substrate analogue.

The crystal structure of the complex formed between phospholipase C (PLC) from Bacillus cereus and inorganic phosphate (Pi), which is an inhibitor, has been determined and refined to 2.1 A resolution. The final R-factor is 19.7%. We have also studied the binding of two other inhibitors, iodide and iodate, to PLC. X-ray data for these two complexes were collected to 2.8 A resolution during the search for heavy-atom derivatives. A series of screening experiments where PLC crystals have been treated with several reaction products and a substrate analogue were carried out to clarify the question of substrate binding. The results have so far been ambiguous but are discussed briefly. Phosphate and iodate are both found to bind to the three metal ions in the protein molecule, suggesting that these ions are involved directly in the catalytic process and thereby identifying the active site. PLC also binds nine iodide ions, eight of which are on the surface of the molecule and of lower occupancy. The ninth blocks the entrance to the active site cleft and is of higher occupancy. Altogether, these results suggest that the substrate, a phospholipid, is associated directly with the metal ions during catalysis.     

X-ray structure of ILL2, an auxin-conjugate amidohydrolase from Arabidopsis thaliana

The plant hormone indole-3-acetic acid (IAA) is the most abundant natural auxin involved in many aspects of plant development and growth. The IAA levels in plants are modulated by a specific group of amidohydrolases from the peptidase M20D family that release the active hormone from its conjugated storage forms. Here, we describe the X-ray crystal structure of IAA-amino acid hydrolase IAA-leucine resistantlike gene 2 (ILL2) from Arabidopsis thaliana at 2.0 A resolution. ILL2 preferentially hydrolyses the auxin-amino acid conjugate N-(indol-3-acetyl)-alanine. The overall structure of ILL2 is reminiscent of dinuclear metallopeptidases from the M20 peptidase family. The structure consists of two domains, a larger catalytic domain with three-layer alpha beta alpha sandwich architecture and aminopeptidase topology and a smaller satellite domain with two-layer alphabeta-sandwich architecture and alpha-beta-plaits topology. The metal-coordinating residues in the active site of ILL2 include a conserved cysteine that clearly distinguishes this protein from previously structurally characterized members of the M20 peptidase family. Modeling of N-(indol-3-acetyl)-alanine into the active site of ILL2 suggests that Leu175 serves as a key determinant for the amino acid side-chain specificity of this enzyme. Furthermore, a hydrophobic pocket nearby the catalytic dimetal center likely recognizes the indolyl moiety of the substrate. Finally, the active site of ILL2 harbors an absolutely conserved glutamate (Glu172), which is well positioned to act as a general acid-base residue. Overall, the structure of ILL2 suggests that this enzyme likely uses a catalytic mechanism that follows the paradigm established for the other enzymes of the M20 peptidase family.     

Functional identification of ligands for a catalytic metal ion in group I introns

Many enzymes use metal ions within their active sites to achieve enormous rate acceleration. Understanding how metal ions mediate catalysis requires elucidation of metal ion interactions with both the enzyme and the substrate(s). The three-dimensional arrangement determined by X-ray crystallography provides a powerful starting point for identifying ground state interactions, but only functional studies can establish and interrogate transition state interactions. The Tetrahymena group I ribozyme is a paradigm for the study of RNA catalysis, and previous work using atomic mutagenesis and quantitative analysis of metal ion rescue behavior identified catalytic metal ions making five contacts with the substrate atoms. Here, we have combined atomic mutagenesis with site-specific phosphorothioate substitutions in the ribozyme backbone to establish transition state ligands on the ribozyme for one of the catalytic metal ions, referred to as M A. We identified the pro-S P oxygen atoms at nucleotides C208, A304, and A306 as ground state ligands for M A, verifying interactions suggested by the Azoarcus crystal structures. We further established that these interactions are present in the chemical transition state, a conclusion that requires functional studies, such as those carried out herein. Elucidating these active site connections is a crucial step toward an in-depth understanding of how specific structural features of the group I intron lead to catalysis.     

The active site of hydroxynitrile lyase from Prunus amygdalus: Modeling studies provide new insights into the mechanism of cyanogenesis

The FAD-dependent hydroxynitrile lyase from almond (Prunus amygdalus, PaHNL) catalyzes the cleavage of R-mandelonitrile into benzaldehyde and hydrocyanic acid. Catalysis of the reverse reaction-the enantiospecific formation of alpha-hydroxynitriles--is now widely utilized in organic syntheses as one of the few industrially relevant examples of enzyme-mediated C-C bond formation. Starting from the recently determined X-ray crystal structure, systematic docking calculations with the natural substrate were used to locate the active site of the enzyme and to identify amino acid residues involved in substrate binding and catalysis. Analysis of the modeled substrate complexes supports an enzymatic mechanism that includes the flavin cofactor as a mere "spectator" of the reaction and relies on general acid/base catalysis by the conserved His-497. Stabilization of the negative charge of the cyanide ion is accomplished by a pronounced positive electrostatic potential at the binding site. PaHNL activity requires the FAD cofactor to be bound in its oxidized form, and calculations of the pKa of enzyme-bound HCN showed that the observed inactivation upon cofactor reduction is largely caused by the reversal of the electrostatic potential within the active site. The suggested mechanism closely resembles the one proposed for the FAD-independent, and structurally unrelated HNL from Hevea brasiliensis. Although the actual amino acid residues involved in the catalytic cycle are completely different in the two enzymes, a common motif for the mechanism of cyanogenesis (general acid/base catalysis plus electrostatic stabilization of the cyanide ion) becomes evident.     

L-aspartase from Escherichia coli: substrate specificity and role of divalent metal ions

The enzyme L-aspartase from Escherichia coli has an absolute specificity for its amino acid substrate. An examination of a wide range of structural analogues of L-aspartic acid did not uncover any alternate substrates for this enzyme. A large number of competitive inhibitors of the enzyme have been characterized, with inhibition constants ranging over 2 orders of magnitude. A divalent metal ion is required for enzyme activity above pH 7, and this requirement is met by many transition and alkali earth metals. The binding stoichiometry has been established to be one metal ion bound per subunit. Paramagnetic relaxation studies have shown that the divalent metal ion binds at the recently discovered activator site on L-aspartase and not at the enzyme active site. Enzyme activators are bound within 5 A of the enzyme-bound divalent metal ion. The activator site is remote from the active site of the enzyme, since the relaxation of inhibitors that bind at the active site is not affected by paramagnetic metal ions bound at the activator site.