The basis for k(cat) impairment in prophospholipase A(2) from the anion-assisted dimer structure

Kinetic results in this paper show that, contrary to earlier reports, pig pancreatic prophospholipase A(2) (proPLA2) does not hydrolyze monodisperse short chain phosphatidylcholine below the critical micelle concentration. ProPLA2 is active on an anionic interface, but at a rate that is decreased by more than 100-fold compared to that of PLA2, the active form. Solution studies show that both proPLA2 and PLA2 bind to an anionic interface and also bind a tetrahedral intermediate mimic at the active site. The 1.5 A resolution crystal structure of the anion-assisted dimer of proPLA2 reported in this paper is compared with the corresponding structure for PLA2 [Pan, Y. H., et al. (2001) Biochemistry 40, 609-617]. As a mimic for the forms bound to the anionic interface, these structures provide insights into the possible structural basis for the impaired chemical step of the zymogen. The proPLA2 dimer contained within one crystallographic asymmetric unit has one molecule of the inhibitor 1-hexadecyl-3-(trifluoroethyl)-sn-glycero-2-phosphomethanol and is bridged by four coplanar sulfate anions. Relative to the structure of PLA2, the subunit contact surface in proPLA2 displays a tilted orientation, an altered mode of inhibitor binding, displacement of a mechanistically significant loop that includes Tyr69, and a critical active site water seen in PLA2 that is not seen in proPLA2. These differences are interpreted to suggest possible origins of the functional differences between the pro and active enzyme at an anionic interface. A structural origin of this difference is discussed in terms of the calcium-coordinated activated water mechanism of the esterolysis reaction. Together, a comparison of the structures of the anion-assisted dimers of PLA2 and proPLA2 not only offers an explanation of why the zymogen form is k(cat)-impaired and binds poorly even to the anionic interface but also supports a mechanism for the activated enzyme that includes a critical second-sphere assisting water bridging His48 and the calcium-coordinated catalytic water.     

Crystal structure of phospholipase A2 complex with the hydrolysis products of platelet activating factor: equilibrium binding of fatty acid and lysophospholipid-ether at the active site may be mutually exclusive

We have solved the 1.55 A crystal structure of the anion-assisted dimer of porcine pancreatic group IB phospholipase A2 (PLA2), complexed with the products of hydrolysis of the substrate platelet activating factor. The dimer contains five coplanar phosphate anions bound at the contact surface between the two PLA2 subunits. This structure parallels a previously reported anion-assisted dimer that mimics the tetrahedral intermediate of PLA2 bound to a substrate interface [Pan, Y. H., et al. (2001) Biochemistry 40, 609-617]. The dimer structure has a molecule of the product acetate bound in subunit A and the other product 1-octadecyl-sn-glycero-3-phosphocholine (LPC-ether) to subunit B. Therefore, this structure is of the two individual product binary complexes and not of a ternary complex with both products in one active site of PLA2. Protein crystals with bound products were only obtained by cocrystallization starting from the initial substrate. In contrast, an alternate crystal form was obtained when PLA2 was cocrystallized with LPC-ether and succinate, and this crystal form did not contain bound products. The product bound structure has acetate positioned in the catalytic site of subunit A such that one of its oxygen atoms is located 3.5 A from the catalytic calcium. Likewise, a longer than typical Ca-to-Gly(32) carbonyl distance of 3.4 A results in a final Ca coordination that is four-coordinate and has distorted geometry. The other oxygen of acetate makes hydrogen bonds with N(delta)(1)-His(48), O(delta)(1)-Asp(49), and the catalytic assisting water (w7). In contrast, the glycerophosphocholine headgroup of LPC-ether in subunit B makes no contacts with calcium or with the catalytic residues His(48) or Asp(49). The tail of the LPC-ether is located near the active site pocket with the last nine carbons of the sn-1- acyl chain refined in two alternate conformations. The remaining atoms of the LPC-ether product have been modeled into the solvent channel but have their occupancies set to zero in the refined model due to disorder. Together, the crystallographic and equilibrium binding results with the two products show that the simultaneous binding of both the products in a single active site is not favored.     

Five coplanar anion binding sites on one face of phospholipase A2: relationship to interface binding

We report the structures of the crystallographic dimer of porcine pancreatic IB phospholipase A(2) (PLA2) with either five sulfate or phosphate anions bound. In each structure, one molecule of a tetrahedral mimic MJ33 [1-hexadecyl-3-(trifluoroethyl)-sn-glycero-2-phosphomethanol] and the five anions are shared between the two subunits of the dimer. The sn-2-phosphate of MJ33 is bound in the active site of one subunit (A), and the alkyl chain extends into the active site slot of the second subunit (B) across the subunit-subunit interface. The two subunits are packed together with a large hydrophobic and desolvated surface buried between them along with the five anions that define a plane. The anions bind by direct contact with two cationic residues (R6 and K10) per subunit and through closer-range H-bonding interactions with other polarizable ligands. These features of the "dimer" suggest that the binding of PLA2 to the anionic groups at the anionic interface may be dominated by coordination through H-bonding with only a partial charge compensation needed. Remarkably, the plane defined by the contact surface is similar to the i-face of the enzyme [Ramirez, F., and Jain, M. K. (1991) Proteins: Struct., Funct., Genet. 9, 229-239], which has been proposed to make contact with the substrate interface for the interfacial catalytic turnover. Additionally, these structures not only offer a view of the active PLA2 complexed to an anionic interface but also provide insight into the environment of the tetrahedral intermediate in the rate-limiting chemical step of the turnover cycle. Taken together, our results offer an atomic-resolution structural view of the i-face interactions of the active form of PLA2 associated to an anionic interface.     

Dihydroorotase from Escherichia coli: loop movement and cooperativity between subunits

Escherichia coli dihydroorotase has been crystallized in the presence of the product, L-dihydroorotate (L-DHO), and the structure refined at 1.9A resolution. The structure confirms that previously reported (PDB entry 1J79), crystallized in the presence of the substrate N-carbamyl-D,L-aspartate (D, L-CA-asp), which had a dimer in the asymmetric unit, with one subunit having the substrate, L-CA-asp bound at the active site and the other having L-DHO. Importantly, no explanation for the unusual structure was given. Our results now show that a loop comprised of residues 105-115 has different conformations in the two subunits. In the case of the L-CA-asp-bound subunit, this loop reaches in toward the active site and makes hydrogen-bonding contact with the bound substrate molecule. For the L-DHO-bound subunit, the loop faces in the opposite direction and forms part of the surface of the protein. Analysis of the kinetics for conversion of L-DHO to L-CA-asp at low concentrations of L-DHO shows positive cooperativity with a Hill coefficient n=1.57(+/-0.13). Communication between subunits in the dimer may occur via cooperative conformational changes of the side-chains of a tripeptide from each subunit: Arg256-His257-Arg258, near the subunit interface.     

Crystal structure of the "cab"-type beta class carbonic anhydrase from the archaeon Methanobacterium thermoautotrophicum

The structure of the "cab"-type beta class carbonic anhydrase from the archaeon Methanobacterium thermoautotrophicum (Cab) has been determined to 2.1-A resolution using the multiwavelength anomalous diffraction phasing technique. Cab exists as a dimer with a subunit fold similar to that observed in "plant"-type beta class carbonic anhydrases. The active site zinc is coordinated by protein ligands Cys(32), His(87), and Cys(90), with the tetrahedral coordination completed by a water molecule. The major difference between plant- and cab-type beta class carbonic anhydrases is in the organization of the hydrophobic pocket. The structure reveals a Hepes buffer molecule bound 8 A away from the active site zinc, which suggests a possible proton transfer pathway from the active site to the solvent.     

Desolvation map of the i-face of phospholipase A2

The changes in the microenvironment of the Trp-3 on the i-face of pig pancreatic IB phospholipase A2 (PLA2) provide a measure of the tight contact (Ramirez and Jain, Protein Sci. 9, 229-239, 1991) with the substrate interface during the processive interfacial turnover. Spectral changes from the single Trp-substituent at position 1, 2, 6, 10, 19, 20, 31, 53, 56 or 87 on the surface of W3F PLA2 are used to probe the Trp-environment. Based on our current understanding only the residue 87 is away from i-face, therefore all other mutants are well suited to report modest differences along the i-face. All Trp-mutants bind tightly to anionic vesicles. Only those with Trp at 1, 2 or 3 near the rim of the active site on the i-face cause significant perturbation of the catalytic functions. Most other Trp-mutants showed < 3-fold change in the interfacial processive turnover rate and the competitive inhibition by MJ33. Binding of calcium to the enzyme in the aqueous phase had modest effect on the Trp-emission intensity. However, on the binding of the enzyme to the interface the fluorescence change is large, and the rate of oxidation of the Trp-substituent with N-bromosuccinimide depends on the location of the Trp-substituent. These results show that the solvation environment of the Trp-substituents on the i-face is shielded in the enzyme bound to the interface. Additional changes are noticeable if the active site of the bound enzyme is also occupied, however, the catalytically inert zymogen of PLA2 (proPLA2) does not show such changes. Significance of these results in relation to the changes in the solvent accessibility and desolvation of the i-face of PLA2 at the interface is discussed.     

Desolvation map of the i-face of phospholipase A2

The changes in the microenvironment of the Trp-3 on the i-face of pig pancreatic IB phospholipase A₂ (PLA2) provide a measure of the tight contact (Ramirez and Jain, Protein Sci. 9, 229-239, 1991) with the substrate interface during the processive interfacial turnover. Spectral changes from the single Trp-substituent at position 1, 2, 6, 10, 19, 20, 31, 53, 56 or 87 on the surface of W3F PLA2 are used to probe the Trp-environment. Based on our current understanding only the residue 87 is away from i-face, therefore all other mutants are well suited to report modest differences along the i-face. All Trp-mutants bind tightly to anionic vesicles. Only those with Trp at 1, 2 or 3 near the rim of the active site on the i-face cause significant perturbation of the catalytic functions. Most other Trp-mutants showed < 3-fold change in the interfacial processive turnover rate and the competitive inhibition by MJ33. Binding of calcium to the enzyme in the aqueous phase had modest effect on the Trp-emission intensity. However, on the binding of the enzyme to the interface the fluorescence change is large, and the rate of oxidation of the Trp-substituent with N-bromosuccinimide depends on the location of the Trp-substituent. These results show that the solvation environment of the Trp-substituents on the i-face is shielded in the enzyme bound to the interface. Additional changes are noticeable if the active site of the bound enzyme is also occupied, however, the catalytically inert zymogen of PLA2 (proPLA2) does not show such changes. Significance of these results in relation to the changes in the solvent accessibility and desolvation of the i-face of PLA2 at the interface is discussed.     

The structural mechanism for half-the-sites reactivity in an enzyme, thymidylate synthase, involves a relay of changes between subunits.

Thymidylate synthase (TS), a half-the-sites reactive enzyme, catalyzes the final step in the de novo biosynthesis of deoxythymidine monophosphate, dTMP, required for DNA replication. The cocrystal structure of TS from Pneumocystis carinii (PcTS), a new drug target for an important pathogen, with its substrate, deoxyuridine monophosphate (dUMP), and a cofactor mimic, CB3717, was determined. The structure, solved at 2.6 A resolution, shows an asymmetric dimer with two molecules of the substrate dUMP bound yet only one molecule of cofactor analogue bound. The structural evidence reveals that upon binding cofactor analogue and forming a covalent bond from the nucleophilic cysteine to the substrate, dUMP, at one active site, PcTS undergoes a conformational change that renders the opposite monomer incapable of forming a covalent bond or binding a molecule of cofactor analogue. The communication pathway between the two active sites is evident, allowing a structural definition of the basis of half-the-sites reactivity for thymidylate synthase and providing an example of such a mechanism for other half-the-sites reactive enzymes.     

Effects of protein-ligand associations on the subunit interactions of phosphofructokinase from B. stearothermophilus

Differences between the crystal structures of inhibitor-bound and uninhibited forms of phosphofructokinase (PFK) from B. stearothermophilus have led to a structural model for allosteric inhibition by phosphoenolpyruvate (PEP) wherein a dimer-dimer interface within the tetrameric enzyme undergoes a quaternary shift. We have developed a labeling and hybridization technique to generate a tetramer with subunits simultaneously containing two different extrinsic fluorophores in known subunit orientations. This construct has been utilized in the examination of the effects of allosteric ligand and substrate binding on the subunit affinities of tetrameric PFK using several biophysical and spectroscopic techniques including 2-photon, dual-channel fluorescence correlation spectroscopy (FCS). We demonstrate that PEP-binding at the allosteric site is sufficient to reduce the affinity of the active site interface from beyond the limits of experimental detection to nanomolar affinity, while conversely strengthening the interface at which it is bound. The reduced interface affinity is specific to inhibitor binding because binding the activator ADP at the same allosteric site causes no reduction in subunit affinity. With inhibitor bound, the weakened subunit affinity has allowed the kinetics of dimer association to be elucidated.     

Interactions between the catalytic centers and subunit interface of triosephosphate isomerase probed by refolding, active site modification, and subunit exchange.

The effects of unfolding, refolding, and hybridization of triosephosphate isomerase (TPI) subunits from different species and subunits which have been specifically modified at the active site have been examined. These effects have been evaluated in terms of changes in catalytic parameters, CD spectra, and susceptibility to denaturation. Dissociation followed by reassociation yields an active dimer but with increased Km, reduced kcat, and increased susceptibility to inactivation and unfolding in denaturants. These data suggest that while the general structure of the refolded dimer is similar to the native enzyme, its complete original structure is not restored. Covalent reaction of the active site Glu165 with the substrate analogue 3-chloroacetol phosphate (CAP) results in dimers with increased susceptibility to unfolding and inactivation by denaturants (i.e. the rates of inactivation and unfolding are (TPICAP)2 greater than (TPI-TPICAP) greater than (TPI)2). These data point to the interactions between the catalytic center and the subunit interface. Subunits of TPI from different species, in spite of structural differences at the subunit interface, hybridized to active heterodimers. Subunit hybridization was random among monomers from different mammals, preferential between yeast and mammalian or avian monomers. Hybridization did not occur between avian and mammalian monomers under these conditions. These data provide information on the elements in the interface of the dimer and the relationship of the catalytic center with the subunit interface.     

Crystal structure of the copper-containing quercetin 2,3-dioxygenase from Aspergillus japonicus

Quercetin 2,3-dioxygenase is a copper-containing enzyme that catalyzes the insertion of molecular oxygen into polyphenolic flavonols. Dioxygenation catalyzed by iron-containing enzymes has been studied extensively, but dioxygenases employing other metal cofactors are poorly understood. We determined the crystal structure of quercetin 2,3-dioxygenase at 1.6 A resolution. The enzyme forms homodimers, which are stabilized by an N-linked heptasaccharide at the dimer interface. The mononuclear type 2 copper center displays two distinct geometries: a distorted tetrahedral coordination, formed by His66, His68, His112, and a water molecule, and a distorted trigonal bipyramidal environment, which additionally comprises Glu73. Manual docking of the substrate quercetin into the active site showed that the different geometries of the copper site might be of catalytic importance.     

Crystallographic structure of phosphofructokinase-2 from Escherichia coli in complex with two ATP molecules. Implications for substrate inhibition

Phosphofructokinase-1 and -2 (Pfk-1 and Pfk-2, respectively) from Escherichia coli belong to different homologous superfamilies. However, in spite of the lack of a common ancestor, they share the ability to catalyze the same reaction and are inhibited by the substrate MgATP. Pfk-2, an ATP-dependent 6-phosphofructokinase member of the ribokinase-like superfamily, is a homodimer of 66 kDa subunits whose oligomerization state is necessary for catalysis and stability. The presence of MgATP favors the tetrameric form of the enzyme. In this work, we describe the structure of Pfk-2 in its inhibited tetrameric form, with each subunit bound to two ATP molecules and two Mg ions. The present structure indicates that substrate inhibition occurs due to the sequential binding of two MgATP molecules per subunit, the first at the usual site occupied by the nucleotide in homologous enzymes and the second at the allosteric site, making a number of direct and Mg-mediated interactions with the first. Two configurations are observed for the second MgATP, one of which involves interactions with Tyr23 from the adjacent subunit in the dimer and the other making an unusual non-Watson-Crick base pairing with the adenine in the substrate ATP. The oligomeric state observed in the crystal is tetrameric, and some of the structural elements involved in the binding of the substrate and allosteric ATPs are also participating in the dimer-dimer interface. This structure also provides the grounds to compare analogous features of the nonhomologous phosphofructokinases from E. coli.     

Structural insights into the second step of RNA-dependent cysteine biosynthesis in archaea: crystal structure of Sep-tRNA:Cys-tRNA synthase from Archaeoglobus fulgidus

In the ancient organisms, methanogenic archaea, lacking the canonical cysteinyl-tRNA synthetase, Cys-tRNA(Cys) is produced by an indirect pathway, in which O-phosphoseryl-tRNA synthetase ligates O-phosphoserine (Sep) to tRNA(Cys) and Sep-tRNA:Cys-tRNA synthase (SepCysS) converts Sep-tRNA(Cys) to Cys-tRNA(Cys). In this study, the crystal structure of SepCysS from Archaeoglobus fulgidus has been determined at 2.4 A resolution. SepCysS forms a dimer, composed of monomers bearing large and small domains. The large domain harbors the seven-stranded beta-sheet, which is typical of the pyridoxal 5'-phosphate (PLP)-dependent enzymes. In the active site, which is located near the dimer interface, PLP is covalently bound to the side-chain of the conserved Lys209. In the proximity of PLP, a sulfate ion is bound by the side-chains of the conserved Arg79, His103, and Tyr104 residues. The active site is located deep within the large, basic cleft to accommodate Sep-tRNA(Cys). On the basis of the surface electrostatic potential, the amino acid residue conservation mapping, the position of the bound sulfate ion, and the substrate amino acid binding manner in other PLP-dependent enzymes, a binding model of Sep-tRNA(Cys) to SepCysS was constructed. One of the three strictly conserved Cys residues (Cys39, Cys42, or Cys247), of one subunit may play a crucial role in the catalysis in the active site of the other subunit.     

Interactions of Trimeresurus flavoviridis phospholipase A2 and its N-terminal octapeptide-removed and p-bromophenacylated derivatives with acridine and anilinonaphthalene dyes

Interactions of dimeric Trimeresurus flavoviridis (the Habu snake) phospholipase A2 (PLA2), des-octapeptide(1-8)-PLA2 (L-fragment) (14% of PLA2 activity), and p-bromophenacyl bromide (BPB)-inactivated PLA2 (BP-PLA2) with dyes, namely, proflavine, 1-anilinonaphthalene-8-sulfonate (Ans), and 2-toluidinylnaphthalene-6-sulfonate (Tns), were investigated. All dyes were bound in a 1:1 molar ratio to the subunit of the proteins. Proflavine was bound most strongly to PLA2 and Ans and Tns were bound to the three proteins with comparable affinities. Capabilities of the dyes for inhibiting alkylation of His-47 of PLA2 with BPB were in the following order: Ans greater than proflavine greater than Tns. Fluorescences of Ans and Tns that were increased in the presence of PLA2 were further greatly enhanced upon the addition of Ca2+, with concomitant formation of the ternary complexes. Ca2+, however, inhibited, competitively or noncompetitively, the bindings of the dyes to PLA2. All dyes were bound to the active site of PLA2 but with different orientations. Inactivation of L-fragment with BPB was inhibited by the dyes in the following order: Tns greater than proflavine approximately Ans. Addition of Ca2+ to the binary complexes formed from L-fragment and Ans or Tns caused no additional enhancement of fluorescence in spite of the formation of the ternary complexes. The active site structures are different between PLA2 and L-fragment, and the N-terminal octapeptide moiety of PLA2 possibly plays a role in maintaining the optimally arranged active site structure of the molecule. Comparison of the data suggests that the N-terminal moieties of PLA2S from snakes of an elapid family and from mammalian pancreas are essential for catalysis of a micellar substrate, whereas those of PLA2S from snakes of a viperid family, such as T. flavoviridis, are not. BP-PLA2 bound Ca2+ and was similar to L-fragment in terms of the fluorescence measurements. It appears that the active site of PLA2 has a space large enough to accommodate p-bromophenacyl, Ans or Tns, and Ca2+ together. Comparison of the emission maxima of Ans and Tns complexed with the three proteins indicated that Tns could be a useful fluorescent probe informing us of the state (disorder) of the active site of PLA2.     

Structural mechanism for glycogen phosphorylase control by phosphorylation and AMP

The crystal structures of activated R state glycogen phosphorylase a (GPa) and R and T state glycogen phosphorylase b (GPb) complexed with AMP have been solved at 2.9 A, 2.9 A and 2.2 A resolution, respectively. The structure of R state GPa is nearly identical to the structure of sulphate-activated R state GPb, except in the region of Ser14, where there is a covalently attached phosphate group in GPa and a non-covalently attached sulphate group in GPb. The contacts made by the N-terminal tail residues in R state GPa at the subunit interface of the functionally active dimer are similar to those observed previously for T state GPa. The quaternary and tertiary structural changes on the T to R transition allow these interactions to be relayed to the catalytic site in R state GPa. The transition from the T state GPb structure to the R state GPa structure results in a change in the N-terminal residues from a poorly ordered extended structure that makes intrasubunit contacts to an ordered coiled conformation that makes intersubunit contacts. The distance between Arg10, the first residue to be located from the N terminus, in R state GPa and T state GPb is 50 A. One of the important subunit-subunit interactions in the dimer molecule involves contacts between the helix alpha 2 and the cap' (residues 35' to 45' that form a loop between the 1st and 2nd alpha helices, alpha 1' and alpha 2' of the other subunit. The prime denotes residues from the other subunit). The interactions made by the N-terminal residues induce structural changes at the cap'/alpha 2 helix interface that lead to the creation of a high-affinity AMP site. The tertiary structural changes at the cap (shifts 1.2 to 2.1 A for residues 35 to 45) are partially compensated by the quaternary structural change so that the overall shifts in these residues after the combined tertiary and quaternary changes are between 0.5 and 1.3 A. AMP binds to R state GPb with at least 100-fold greater affinity and exhibits four additional hydrogen bonds, stronger ionic interactions and more extensive van der Waals' interactions with 116 A2 greater solvent accessible surface area buried compared with AMP bound to T state GPb.(ABSTRACT TRUNCATED AT 400 WORDS)     

Long-range interactions in the dimer interface of ornithine decarboxylase are important for enzyme function.

Ornithine decarboxylase (ODC) is a pyridoxal 5'-phosphate dependent enzyme that catalyzes the first committed step in the biosynthesis of polyamines. ODC is a proven drug target for the treatment of African sleeping sickness. The enzyme is an obligate homodimer, and the two identical active sites are formed at the dimer interface. Alanine scanning mutagenesis of dimer interface residues in Trypanosoma brucei ODC was undertaken to determine the energetic contribution of these residues to subunit association. Twenty-three mutant enzymes were analyzed by analytical ultracentrifugation, and none of the mutations were found to cause a greater than 1 kcal/mol decrease in dimer stability. These data suggest that the energetics of the interaction may be distributed across the interface. Most significantly, many of the mutations had large effects (DeltaDeltaG kcat/Km > 2.5 kcal/mol) on the catalytic efficiency of the enzyme. Residues that affected activity included those in or near the substrate binding site but also a number of residues that are distant (15-20 A) from this site. These data provide evidence that long-range energetic coupling of interface residues to the active site is essential for enzyme function, even though structural changes upon ligand binding to wild-type ODC are limited to local conformational changes in the active site. The ODC dimer interface appears to be optimized for catalytic function and not for dimer stability. Thus, small molecules directed to the ODC interfaces could impact biological function without having to overcome the difficult energetic barrier of dissociating the interacting partners.     

Is dimerization required for the catalytic activity of bacterial biotin carboxylase?

Acetyl-coenzyme A carboxylases (ACCs) have crucial roles in fatty acid metabolism. The biotin carboxylase (BC) subunit of Escherichia coli ACC is believed to be active only as a dimer, although the crystal structure shows that the active site of each monomer is 25 A from the dimer interface. We report here biochemical, biophysical, and structural characterizations of BC carrying single-site mutations in the dimer interface. Our studies demonstrate that two of the mutants, R19E and E23R, are monomeric in solution but have only a 3-fold loss in catalytic activity. The crystal structures of the E23R and F363A mutants show that they can still form the correct dimer at high concentrations. Our data suggest that dimerization is not an absolute requirement for the catalytic activity of the E. coli BC subunit, and we propose a new model for the molecular mechanism of action for BC in multisubunit and multidomain ACCs.     

Identification of an allosteric anion-binding site on O-acetylserine sulfhydrylase: structure of the enzyme with chloride bound

A new crystal structure of O-acetylserine sulfhydrylase (OASS) has been solved with chloride bound at an allosteric site and sulfate bound at the active site. The bound anions result in a new "inhibited" conformation, that differs from the "open" native or "closed" external aldimine conformations. The allosteric site is located at the OASS dimer interface. The new inhibited structure involves a change in the position of the "moveable domain" (residues 87-131) to a location that differs from that in the open or closed forms. Formation of the external aldimine with substrate is stabilized by interaction of the alpha-carboxyl group of the substrate with a substrate-binding loop that is part of the moveable domain. The inhibited conformation prevents the substrate-binding loop from interacting with the alpha-carboxyl group, and hinders formation of the external Schiff base and thus subsequent chemistry. Chloride may be an analog of sulfide, the physiological inhibitor. Finally, these results suggest that OASS represents a new class of PLP-dependent enzymes that is regulated by small anions.     

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.     

Effects of dimerization of Serratia marcescens endonuclease on water dynamics

The dynamics and structure of Serratia marcescens endonuclease and its neighboring solvent are investigated by molecular dynamics (MD). Comparisons are made with structural and biochemical experiments. The dimer form is physiologic and functions more processively than the monomer. We previously found a channel formed by connected clusters of waters from the active site to the dimer interface. Here, we show that dimerization clearly changes correlations in the water structure and dynamics in the active site not seen in the monomer. Our results indicate that water at the active sites of the dimer is less affected compared with bulk solvent than in the monomer where it has much slower characteristic relaxation times. Given that water is a required participant in the reaction, this gives a clear advantage to dimerization in the absence of an apparent ability to use both active sites simultaneously.