Phosphate Binding Site Research Articles

Mechanism of the phosphatase component of Clostridium thermocellum polynucleotide kinase-phosphatase.

Polynucleotide kinase-phosphatase (Pnkp) from Clostridium thermocellum catalyzes ATP-dependent phosphorylation of 5'-OH termini of DNA or RNA polynucleotides and Ni(2+)/Mn(2+)-dependent dephosphorylation of 2',3' cyclic phosphate, 2'-phosphate, and 3'-phosphate ribonucleotides. CthPnkp is an 870-amino-acid polypeptide composed of three domains: an N-terminal module similar to bacteriophage T4 polynucleotide kinase, a central module that resembles the dinuclear metallo-phosphoesterase superfamily, and a C-terminal ligase-like adenylyltransferase domain. Here we conducted a mutational analysis of CthPnkp that identified 11 residues required for Ni(2+)-dependent phosphatase activity with 2'-AMP and 3'-AMP. Eight of the 11 CthPnkp side chains were also required for Ni(2+)-dependent hydrolysis of p-nitrophenyl phosphate. The ensemble of essential side chains includes the conserved counterparts (Asp187, His189, Asp233, Arg237, Asn263, His264, His323, His376, and Asp392 in CthPnkp) of all of the amino acids that form the dinuclear metal-binding site and the phosphate-binding site of bacteriophage lambda phosphatase. Three residues (Asp236, His264, and Arg237) required for activity with 2'-AMP or 3'-AMP were dispensable for Ni(2+)-dependent hydrolysis of p-nitrophenyl phosphate. Our findings, together with available structural information, provide fresh insights to the metallophosphoesterase mechanism, including the roles of His264 and Asp236 in proton donation to the leaving group. Deletion analysis defined an autonomous phosphatase domain, CthPnkp-(171-424).

Crystal structures of a phosphotransacetylase from Bacillus subtilis and its complex with acetyl phosphate

Phosphotransacetylase (Pta) [EC 2.3.1.8] plays a major role in acetate metabolism by catalyzing the reversible transfer of the acetyl group between coenzyme A (CoA) and orthophosphate: CH(3)COSCoA+HPO(4)(2-)<-->CH(3)COOPO(3)(2-) +CoASH. In this study, we report the crystal structures of Pta from Bacillus subtilis at 2.75 A resolution and its complex with acetyl phosphate, one of its substrates, at 2.85 A resolution. In addition, the Pta activity of the enzyme has been assayed. The enzyme folds into an alpha/beta architecture with two domains separated by a prominent cleft, very similar to two other known Pta structures. The enzyme-acetyl phosphate complex structure reveals a few potential substrate binding sites. Two of them are located in the middle of the interdomain cleft: each one is surrounded by a region of strictly and highly conserved residues. High structural similarities are found with 4-hydroxythreonine-4-phosphate dehydrogenase (PdxA), and isocitrate and isopropylmalate dehydrogenases, all of which utilize NADP+ as their cofactor, which binds in the interdomain cleft. Their substrate binding sites are close to the acetyl phosphate binding sites of Pta in the cleft as well. These results suggest that the CoA is likely to bind to the interdomain cleft of Pta in a similar way as NADP+ binds to the other three enzymes.

Glucose 6-Phosphate Release of Wild-type and Mutant Human Brain Hexokinases from Mitochondria

One molecule of glucose 6-phosphate inhibits brain hexokinase (HKI) with high affinity by binding to either one of two sites located in distinct halves of the enzyme. In addition to potent inhibition, glucose 6-phosphate releases HKI from the outer leaflet of mitochondria; however, the site of glucose 6-phosphate association responsible for the release of HKI is unclear. The incorporation of a C-terminal polyhistidine tag on HKI facilitates the rapid purification of recombinant enzyme from Escherichia coli. The tagged construct has N-formyl methionine as its first residue and has mitochondrial association properties comparable with native brain hexokinases. Release of wild-type and mutant hexokinases from mitochondria by glucose 6-phosphate follow equilibrium models, which explain the release phenomenon as the repartitioning of ligand-bound HKI between solution and the membrane. Mutations that block the binding of glucose 6-phosphate to the C-terminal half of HKI have little or no effect on the glucose 6-phosphate release. In contrast, mutations that block glucose 6-phosphate binding to the N-terminal half require approximately 7-fold higher concentrations of glucose 6-phosphate for the release of HKI. Results here implicate a primary role for the glucose 6-phosphate binding site at the N-terminal half of HKI in the release mechanism.

Novel multisubstrate inhibitors of mammalian purine nucleoside phosphorylase

In an effort to develop potent multisubstrate-analog inhibitors of purine nucleoside phosphorylase (PNP), three nucleoside phosphonates were designed utilizing structural information from the previously reported structures of complexes of bovine PNP with substrates and products. The nucleoside phosphonates contain an acetal linkage at the O2' and O3' positions and a two-C-atom spacer between the ribose and phosphate moieties. The linkage enables the compounds to simultaneously occupy the purine-, ribose- and phosphate-binding sites. The chemical syntheses, inhibition profiles and structural characterization of these novel multisubstrate analog inhibitors with bovine PNP are described.

Reversible reaction of 5,5′-dithiobis(2-nitrobenzoate) with the hemoglobins of the domestic cat: Acetylation of NH 3+ terminal group of the β chain transforms the complex pH dependence of the forward apparent second order rate constant to a simple form

We demonstrate kinetically that the reaction of 5,5′-dithiobis(2-nitrobenzoate) with the CysF9[93]β sulfhydryl group of domestic cat hemoglobins is a reversible process. In the major hemoglobin, in which the NH 3 + terminal group of GlyNA1[1]β is free, k f, the apparent forward second order rate constant, has a complex pH dependence profile. In the minor hemoglobin, the NH 3 + terminal group of SerNA1[1]β is acetylated, and the pH dependence profile of k f is simple. These results support the proposal that the positively charged groups at the organic phosphate binding site are electrostatically linked to CysF9[93]β. Quantitative analyses of the complex profiles enabled us to estimate pK as of 7.47 ± 0.3; 6.53 ± 0.03 and 8.49 ± 0.3 for GlyNA1[1]β, HisH21[143]β and other histidines within 2 nm of the sulfhydryl, and CysF9[93]β, respectively, of the major hemoglobin. Analyses of the simple profiles gave pK as of 6.33 ± 0.17 and 8.54 ± 0.5 for HisH21[143]β and other histidines within a distance of 2 nm of the sulfhydryl, and CysF9[93]β of the minor hemoglobin, respectively.

Structural Basis of the Cks1-Dependent Recognition of p27 Kip1 by the SCF Skp2 Ubiquitin Ligase

The ubiquitin-mediated proteolysis of the Cdk2 inhibitor p27(Kip1) plays a central role in cell cycle progression, and enhanced degradation of p27(Kip1) is associated with many common cancers. Proteolysis of p27(Kip1) is triggered by Thr187 phosphorylation, which leads to the binding of the SCF(Skp2) (Skp1-Cul1-Rbx1-Skp2) ubiquitin ligase complex. Unlike other known SCF substrates, p27(Kip1) ubiquitination also requires the accessory protein Cks1. The crystal structure of the Skp1-Skp2-Cks1 complex bound to a p27(Kip1) phosphopeptide shows that Cks1 binds to the leucine-rich repeat (LRR) domain and C-terminal tail of Skp2, whereas p27(Kip1) binds to both Cks1 and Skp2. The phosphorylated Thr187 side chain of p27(Kip1) is recognized by a Cks1 phosphate binding site, whereas the side chain of an invariant Glu185 inserts into the interface between Skp2 and Cks1, interacting with both. The structure and biochemical data support the proposed model that Cdk2-cyclin A contributes to the recruitment of p27(Kip1) to the SCF(Skp2)-Cks1 complex.

Two classes of p38α MAP kinase inhibitors having a common diphenylether core but exhibiting divergent binding modes

Two new classes of diphenylether inhibitors of p38α MAP kinase are described. Both chemical classes are based on a common diphenylether core that is identified by simulated fragment annealing as one of the most favored chemotypes within a prominent hydrophobic pocket of the p38α ATP-binding site. In the fully elaborated molecules, the diphenylether moiety acts as an anchor occupying the deep pocket, while polar extensions make specific interactions with either the adenine binding site or the phosphate binding site of ATP. The synthesis, crystallographic analysis, and biological activity of these p38α inhibitors are discussed.

Structures for the Potential Drug Target Purine Nucleoside Phosphorylase from Schistosoma mansoni Causal Agent of Schistosomiasis

Despite the availability of effective chemotherapy, schistosomiasis continues to be one of the major parasitic infections to affect the human population worldwide. Currently, little is known of the structural biology of the parasites that are responsible for the disease and few attempts have been made to develop second generation drugs, which may become essential if resistance to those currently available becomes an issue. Here, we describe three crystal structures for the enzyme purine nucleoside phosphorylase (PNP) from Schistosoma mansoni, a component of the purine salvage pathway. PNP is known to be essential for the recovery of purine bases and nucleosides in schistosomes, due to an absence of the enzymes for de novo synthesis, making it a sensitive point in the parasite's metabolism. In all three structures reported here, acetate occupies part of the base-binding site and is directly bound to the conserved glutamic acid at position 203. One of the structures presents the crystallization additive sulfobetaine 195 (NDSB195) occupying simultaneously the ribose and phosphate binding sites, whilst a second presents only phosphate in the latter. The observation of sulfobetaine specifically bound to the protein active site was unexpected and is unique to this structure as far as we are aware. Considerable flexibility is observed in the active site, principally due to variable structural disorder in the regions centered on residues 64 and 260. This conformational plasticity extends to the way in which both NDSB195 and phosphate bind to the individual monomers of the trimeric structure reported here. Differences between the parasite and human enzymes are limited principally to the base-binding site, where the substitution of V245 in the mammalian enzymes by S247 introduces additional hydrogen bonding potential to the site. This is satisfied in the structures described here by a water molecule whose presence is normally observed only in complexes with 6-oxopurines. Residue Y202, which replaces F200 in human PNP, is able to reach over the ribose-binding site to interact with H259 and is predicted to form an additional hydrogen bond with the 5' hydroxyl of nucleoside substrates.

Design, synthesis, and biochemical evaluation of 1,5,6,7-tetrahydro-6,7-dioxo-9-D-ribitylaminolumazines bearing alkyl phosphate substituents as inhibitors of lumazine synthase and riboflavin synthase.

The last two steps in the biosynthesis of riboflavin, an essential metabolite that is involved in electron transport, are catalyzed by lumazine synthase and riboflavin synthase. To obtain structural probes and inhibitors of these two enzymes, two ribityllumazinediones bearing alkyl phosphate substituents were synthesized. The synthesis involved the generation of the ribityl side chain, the phosphate side chain, and the lumazine system in protected form, followed by the simultaneous removal of three different types of protecting groups. The products were designed as intermediate analogue inhibitors of lumazine synthase that would bind to its phosphate-binding site as well as its lumazine binding site. Both compounds were found to be effective inhibitors of Bacillus subtilislumazine synthase as well as Escherichia coli riboflavin synthase. Molecular modeling of the binding of one of the two compounds provided a structural explanation for how these compounds are able to effectively inhibit both enzymes. In phosphate-free buffer, the phosphate moieties of the inhibitors were found to contribute positively to their binding to Mycobacterium tuberculosis lumazine synthase, resulting in very potent inhibitors with Ki values in the low nanomolar range. The additional carbonyl in the dioxolumazine system versus the purinetrione system was found to make a positive contribution to its binding to E. coli riboflavin synthase.

Two sites for GTP binding in cathodic haemoglobins from Anguilliformes

Cathodic haemoglobins of four species of anguilliform fish were characterized from a functional point of view, with special regard to the interaction with their physiological effectors. A series of oxygen-binding experiments at increasing GTP concentrations was carried out in order to compare GTP-binding activities in the absence and presence of saturating amounts of chloride. The results indicated that the cathodic haemoglobin of three species ( Anguilla anguilla, Conger conger and Muraena helena) do have two sites for GTP-binding. In the absence of chloride, the two sites cannot be discriminated, whereas in the presence of chloride, a competition between the two anions occurred for the second GTP-binding site. The cathodic haemoglobin of Gymnothorax unicolor, which showed lower GTP sensitivity than the other haemoglobins examined, displayed only one GTP-binding site. The presence of an additional phosphate-binding site is not exceptional, although the way haemoglobin interacts with the two organic phosphate molecules may differ among species. This property may provide an auxiliary means of haemoglobin modulation for species that inhabit environments where oxygen availability is highly variable and haemoglobin-oxygen affinity needs to be modulated to different extents in order to satisfy physiological oxygen requirements.

A New Arrangement of (β/α)8 Barrels in the Synthase Subunit of PLP Synthase

Pyridoxal 5'-phosphate (PLP, vitamin B6), a cofactor in many enzymatic reactions, has two distinct biosynthetic routes, which do not coexist in any organism. Two proteins, known as PdxS and PdxT, together form a PLP synthase in plants, fungi, archaea, and some eubacteria. PLP synthase is a heteromeric glutamine amidotransferase in which PdxT produces ammonia from glutamine and PdxS combines ammonia with five- and three-carbon phosphosugars to form PLP. In the 2.2-A crystal structure, PdxS is a cylindrical dodecamer of subunits having the classic (beta/alpha)8 barrel fold. PdxS subunits form two hexameric rings with the active sites positioned on the inside. The hexamer and dodecamer forms coexist in solution. A novel phosphate-binding site is suggested by bound sulfate. The sulfate and another bound molecule, methyl pentanediol, were used to model the substrate ribulose 5-phosphate, and to propose catalytic roles for residues in the active site. The distribution of conserved surfaces in the PdxS dodecamer was used to predict a docking site for the glutaminase partner, PdxT.

Modulation of Charge in the Phosphate Binding Site of Escherichia coli ATP Synthase

This paper presents a study of the role of positive charge in the P(i) binding site of Escherichia coli ATP synthase, the enzyme responsible for ATP-driven proton extrusion and ATP synthesis by oxidative phosphorylation. Arginine residues are known to occur with high propensity in P(i) binding sites of proteins generally and in the P(i) binding site of the betaE catalytic site of ATP synthase specifically. Removal of natural betaArg-246 (betaR246A mutant) abrogates P(i) binding; restoration of P(i) binding was achieved by mutagenesis of either residue betaAsn-243 or alphaPhe-291 to Arg. Both residues are located in the P(i) binding site close to betaArg-246 in x-ray structures. Insertion of one extra Arg at beta-243 or alpha-291 in presence of betaArg-246 retained P(i) binding, but insertion of two extra Arg, at both positions simultaneously, abrogated it. Transition state stabilization was measured using phosphate analogs fluoroaluminate and fluoroscandium. Removal of betaArg-246 in betaR246A caused almost complete loss of transition state stabilization, but partial rescue was achieved in betaN243R/betaR246A and alphaF291R/betaR246A. BetaArg-243 or alphaArg-291 in presence of betaArg-246 was less effective; the combination of alphaF291R/betaN243R with natural betaArg-246 was just as detrimental as betaR246A. The data demonstrate that electrostatic interaction is an important component of initial P(i) binding in catalytic site betaE and later at the transition state complex. However, since none of the mutants showed significant function in growth tests, ATP-driven proton pumping, or ATPase activity assays, it is apparent that specific stereochemical interactions of catalytic site Arg residues are paramount.

An end-healing enzyme from Clostridium thermocellum with 5′ kinase, 2′,3′ phosphatase, and adenylyltransferase activities

We identify and characterize an end-healing enzyme, CthPnkp, from Clostridium thermocellum that catalyzes the phosphorylation of 5'-OH termini of DNA or RNA polynucleotides and the dephosphorylation of 2',3' cyclic phosphate, 2'-phosphate, and 3'-phosphate ribonucleotides. CthPnkp also catalyzes an autoadenylylation reaction via a polynucleotide ligase-type mechanism. These characteristics are consistent with a role in end-healing during RNA or DNA repair. CthPnkp is a homodimer of an 870-amino-acid polypeptide composed of three catalytic domains: an N-terminal module that resembles the polynucleotide kinase domain of bacteriophage T4 Pnkp, a central metal-dependent phosphoesterase module, and a C-terminal module that resembles the nucleotidyl transferase domain of polynucleotide ligases. The distinctive feature of CthPnkp vis-à-vis known RNA repair enzymes is that its 3' end modification component belongs to the calcineurin-type phosphatase superfamily. It contains putative counterparts of the amino acids that form the dinuclear metal-binding site and the phosphate-binding site of bacteriophage lambda phosphatase. As with lambda phosphatase, the 2',3' cAMP phosphatase activity of CthPnkp is specifically dependent on nickel or manganese. We identify homologs of CthPnkp in other bacterial proteomes.

Lead biosorption study with Rhizopus arrhizus using a metal-based titration technique

Acid–base and metal-based potentiometric titration methods were used to analyze sorption mechanisms of lead by Rhizopus arrhizus fungal biomass. Biosorption was not considered globally but as the result of successive sorption reactions on various binding sites with different selectivities. Precipitation occurred rapidly when lead concentration increased. Lead was sorbed essentially by carboxylic groups and by phosphates and sulfonates (less abundant) of the organic matter. The lead affinity to carboxylic, sulfonate and phosphate binding sites depended on the association coefficient with proton or counter-ion and on the spatial distribution of the surface sites promoting the formation of mono- or bi-dentate complexes. Chemical bonds and binding sites were confirmed using microscopic and spectroscopic techniques (IR, MET-EDAX). It appeared that although the total organic acidity was reached, number of ionized and free carboxylic groups were not involved in lead sorption reactions. In spite of lead speciation in the solution, surface micro-precipitation was observed and the two processes, surface adsorption and micro-precipitation, are sequential and possibly overlapping. At low concentrations ( < 10 −6 M ) adsorption is the dominant phenomenon and beyond ( > 10 −5 M ) surface clusters appeared before the predicted solution precipitation phenomenon.

Mutagenesis at Two Distinct Phosphate-Binding Sites Unravels Their Differential Roles in Regulation of Rubisco Activation and Catalysis

Orthophosphate (P(i)) has two antagonistic effects on ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), stimulation of activation and inhibition of catalysis by competition with the substrate RuBP. The enzyme binds P(i) at three distinct sites, two within the catalytic site (where 1P and 5P of ribulose 1,5-bisphosphate [RuBP] bind), and the third at the latch site (a positively charged pocket involved in active-site closure during catalysis). We examined the role of the latch and 5P sites in regulation of Rubisco activation and catalysis by introducing specific mutations in the enzyme of the cyanobacterium Synechocystis sp. strain PCC 6803. Whereas mutations at both sites abolished the P(i)-stimulated Rubisco activation, substitution of residues at the 5P site, but not at the latch site, affected the P(i) inhibition of Rubisco catalysis. Although some of these mutations substantially reduced the catalytic turnover of Rubisco and increased the K(m)(RuBP), they had little to moderate effect on the rate of photosynthesis and no effect on photoautotrophic growth. These findings suggest that in cyanobacteria, Rubisco does not limit photosynthesis to the extent previously estimated. These results indicate that both the latch and 5P sites participate in regulation of Rubisco activation, whereas P(i) binding only at the 5P site inhibits catalysis in a competitive manner.

High-throughput kinase assays with protein substrates using fluorescent polymer superquenching

BackgroundHigh-throughput screening is used by the pharmaceutical industry for identifying lead compounds that interact with targets of pharmacological interest. Because of the key role that aberrant regulation of protein phosphorylation plays in diseases such as cancer, diabetes and hypertension, kinases have become one of the main drug targets. With the exception of antibody-based assays, methods to screen for specific kinase activity are generally restricted to the use of small synthetic peptides as substrates. However, the use of natural protein substrates has the advantage that potential inhibitors can be detected that affect enzyme activity by binding to a site other than the catalytic site. We have previously reported a non-radioactive and non-antibody-based fluorescence quench assay for detection of phosphorylation or dephosphorylation using synthetic peptide substrates. The aim of this work is to develop an assay for detection of phosphorylation of chemically unmodified proteins based on this polymer superquenching platform.ResultsUsing a modified QTL Lightspeed™ assay, phosphorylation of native protein was quantified by the interaction of the phosphorylated proteins with metal-ion coordinating groups co-located with fluorescent polymer deposited onto microspheres. The binding of phospho-protein inhibits a dye-labeled "tracer" peptide from associating to the phosphate-binding sites present on the fluorescent microspheres. The resulting inhibition of quench generates a "turn on" assay, in which the signal correlates with the phosphorylation of the substrate. The assay was tested on three different proteins: Myelin Basic Protein (MBP), Histone H1 and Phosphorylated heat- and acid-stable protein (PHAS-1). Phosphorylation of the proteins was detected by Protein Kinase Cα (PKCα) and by the Interleukin -1 Receptor-associated Kinase 4 (IRAK4). Enzyme inhibition yielded IC50 values that were comparable to those obtained using peptide substrates. Statistical parameters that are used in the high-throughput community to determine assay robustness (Z'-value) demonstrate the suitability of this format for high-throughput screening applications for detection of inhibitors of enzyme activity.ConclusionThe QTL Lightspeed™ protein detection system provides a simple mix and measure "turn on" assay for the detection of kinase activity using natural protein substrates. The platform is robust and allows for identification of inhibitors of kinase activity.

Crystal Structure of Baculovirus RNA Triphosphatase Complexed with Phosphate

Baculovirus RNA 5'-triphosphatase (BVP) exemplifies a family of RNA-specific cysteine phosphatases that includes the RNA triphosphatase domains of metazoan and plant mRNA capping enzymes. Here we report the crystal structure of BVP in a phosphate-bound state at 1.5 A resolution. BVP adopts the characteristic cysteine-phosphatase alpha/beta fold and binds two phosphate ions in the active site region, one of which is proposed to mimic the phosphate of the product complex after hydrolysis of the covalent phosphoenzyme intermediate. The crystal structure highlights the role of backbone amides and side chains of the P-loop motif (118)HCTHGXNRT(126) in binding the cleavable phosphate and stabilizing the transition state. Comparison of the BVP structure to the apoenzyme of mammalian RNA triphosphatase reveals a concerted movement of the Arg-125 side chain (to engage the phosphate directly) and closure of an associated surface loop over the phosphate in the active site. The structure highlights a direct catalytic role of Asn-124, which is the signature P-loop residue of the RNA triphosphatase family and a likely determinant of the specificity of BVP for hydrolysis of phosphoanhydride linkages.

Physicochemical Characterization of Casein Phosphopeptide-Amorphous Calcium Phosphate Nanocomplexes

Milk caseins stabilize calcium and phosphate ions and make them available to the neonate. Tryptic digestion of the caseins yields phosphopeptides from their polar N-terminal regions that contain clusters of phosphorylated seryl residues. These phosphoseryl clusters have been hypothesized to be responsible for the interaction between the caseins and calcium phosphate that lead to the formation of casein micelles. The casein phosphopeptides stabilize calcium and phosphate ions through the formation of complexes. The calcium phosphate in these complexes is biologically available for intestinal absorption and remineralization of subsurface lesions in tooth enamel. We have studied the structure of the complexes formed by the casein phosphopeptides with calcium phosphate using a range of physicochemical techniques including x-ray powder diffraction, scanning electron microscopy, transmission electron microscopy, and equilibrium binding analyses. The amorphous nature of the calcium phosphate phase was confirmed by two independent methods: x-ray powder diffraction and selected area diffraction. In solution, the ion activity product of a basic amorphous calcium phosphate phase was the only ion product that was a function of bound phosphate independent of pH, consistent with basic amorphous calcium phosphate being the phase stabilized by the casein phosphopeptides. Detailed investigations of calcium and calcium phosphate binding using a library of synthetic homologues and analogues of the casein phosphopeptides have revealed that although the fully phosphorylated seryl-cluster motif is pivotal for the interaction with calcium and phosphate, other factors are also important. In particular, calcium binding and calcium phosphate stabilization by the peptides was influenced by peptide net charge, length, and sequence.

Kinetic Analysis of the l-Ornithine Transcarbamoylase from Pseudomonas savastanoi pv. phaseolicola That Is Resistant to the Transition State Analogue (R)-Nδ-(N‘-Sulfodiaminophosphinyl)-l-ornithine

(R)-N(delta)-(N'-Sulfodiaminophosphinyl)-L-ornithine (PSorn) is the active component of a phytotoxin, called phaseolotoxin, produced by Pseudomonas savastanoi pv. phaseolicola. PSorn acts as a potent transition state (TS) inhibitor of ornithine transcarbamoylase (OTCase, E.C. 2.1.3.3) that binds to the OTCase from Escherichia coli (ARGI) with a dissociation constant of 1.6 pM. While inhibition of OTCase can lead to arginine auxotrophy, P. savastanoi pv. phaseolicola is able to synthesize toxin while growing on minimal medium. This is achieved by the expression during toxin production of a second gene encoding OTCase activity that is not inhibited by PSorn (ROTCase). ROTCase is orthologous to other OTCases, but it has substitutions to key conserved amino acids, particularly to those around the carbamoyl phosphate (CP) binding site and in the ornithine binding "SMG" loop. This suggests that the topology of the CP binding site and the closure of the SMG loop may be different in ROTCase. Steady-state kinetics indicate that ROTCase has an ordered mechanism, and the (13)C kinetic isotope effect (IE) in CP indicates that it is the first substrate to bind. However, unlike other OTCases, there is a random element to the mechanism since the second substrate ornithine (Orn) was unable to completely suppress the IE to unity. The most striking difference with ROTCase is the reduction of k(cat) to between 1% and 2% of other OTCases. This is consistent with the large IE that ROTCase exhibits (3.4%) at near-zero Orn. These results suggest that the chemistry of the reaction is rate limiting for ROTCase. ROTCase has a substrate and inhibitor profile similar to that of other OTCases. The CP binding affinity of ROTCase is diminished when compared with that observed from ARGI, and inhibitors that compete with the CP binding site have K(i) values at least 10-fold higher for ROTCase than for ARGI. Arsenate did not inhibit ROTCase, and bisubstrate and dead-end inhibitors are less effective inhibitors of ROTCase than ARGI. These data suggest that PSorn is unable to bind tightly to either the apo or activated forms of ROTCase at the expense of CP binding and reduced k(cat).

Isolation and characterization of a D‐cysteine desulfhydrase protein from Arabidopsis thaliana

In several organisms D-cysteine desulfhydrase (D-CDes) activity (EC 4.1.99.4) was measured; this enzyme decomposes D-cysteine into pyruvate, H2S, and NH3. A gene encoding a putative D-CDes protein was identified in Arabidopsis thaliana (L) Heynh. based on high homology to an Escherichia coli protein called YedO that has D-CDes activity. The deduced Arabidopsis protein consists of 401 amino acids and has a molecular mass of 43.9 kDa. It contains a pyridoxal-5'-phosphate binding site. The purified recombinant mature protein had a Km for D-cysteine of 0.25 mm. Only D-cysteine but not L-cysteine was converted by D-CDes to pyruvate, H2S, and NH3. The activity was inhibited by aminooxy acetic acid and hydroxylamine, inhibitors specific for pyridoxal-5'-phosphate dependent proteins, at low micromolar concentrations. The protein did not exhibit 1-aminocyclopropane-1-carboxylate deaminase activity (EC 3.5.99.7) as homologous bacterial proteins. Western blot analysis of isolated organelles and localization studies using fusion constructs with the green fluorescent protein indicated an intracellular localization of the nuclear encoded D-CDes protein in the mitochondria. D-CDes RNA levels increased with proceeding development of Arabidopsis but decreased in senescent plants; D-CDes protein levels remained almost unchanged in the same plants whereas specific D-CDes activity was highest in senescent plants. In plants grown in a 12-h light/12-h dark rhythm D-CDes RNA levels were highest in the dark, whereas protein levels and enzyme activity were lower in the dark period than in the light indicating post-translational regulation. Plants grown under low sulfate concentration showed an accumulation of D-CDes RNA and increased protein levels, the D-CDes activity was almost unchanged. Putative in vivo functions of the Arabidopsisd-CDes protein are discussed.