Structure and mutational analysis of a plant mitochondrial nucleoside diphosphate kinase. Identification of residues involved in serine phosphorylation and oligomerization

We report the first crystal structure of a plant (Pisum sativum L. cv Oregon sugarpod) mitochondrial nucleoside diphosphate kinase. Similar to other eukaryotic nucleoside diphosphate kinases, the plant enzyme is a hexamer; the six monomers in the asymmetric unit are arranged as trimers of dimers. Different functions of the kinase have been correlated with the oligomeric structure and the phosphorylation of Ser residues. We show that the occurrence of Ser autophosphorylation depends on enzymatic activity. The mutation of the strictly conserved Ser-119 to Ala reduced the Ser phosphorylation to about one-half of that observed in wild type with only a modest change of enzyme activity. We also show that mutating another strictly conserved Ser, Ser-69, to Ala reduces the enzyme activity to 6% and 14% of wild-type using dCDP and dTDP as acceptors, respectively. Changes in the oligomerization pattern of the S69A mutant were observed by cross-linking experiments. A reduction in trimer formation and a change in the dimer interaction could be detected with a concomitant increase of tetramers. We conclude that the S69 mutant is involved in the stabilization of the oligomeric state of this plant nucleoside diphosphate kinase.     

Activation of factor V during intrinsic and extrinsic coagulation. Inhibition by heparin, hirudin and D-Phe-Pro-Arg-Ch2Cl.

Ser130, Asp131 and Asn132 ('SDN') are highly conserved residues in class A beta-lactamases forming one wall of the active-site cavity. All three residues of the SDN loop in Streptomyces albus G beta-lactamase were modified by site-directed mutagenesis. The mutant proteins were expressed in Streptomyces lividans, purified from culture supernatants and their kinetic parameters were determined for several substrates. Ser130 was substituted by Asn, Ala and Gly. The first modification yielded an almost totally inactive protein, whereas the smaller-side-chain mutants (A and G) retained some activity, but were less stable than the wild-type enzyme. Ser130 might thus be involved in maintaining the structure of the active-site cavity. Mutations of Asp131 into Glu and Gly proved to be highly detrimental to enzyme stability, reflecting significant structural perturbations. Mutation of Asn132 into Ala resulted in a dramatically decreased enzymic activity (more than 100-fold) especially toward cephalosporin substrates, kcat. being the most affected parameter, which would indicate a role of Asn132 in transition-state stabilization rather than in ground-state binding. Comparison of the N132A and the previously described N132S mutant enzymes underline the importance of an H-bond-forming residue at position 132 for the catalytic process.     

Use of Ramachandran Plot for Increasing Thermal Stability of Bacterial Formate Dehydrogenase

From analysis of Ramachandran plot for NAD+-dependent formate dehydrogenase from the methylotrophic bacterium Pseudomonas sp. 101 (FDH, EC 1.2.1.2), five amino acid residues with non-optimal values phi and psi have been located in beta- and pi-turns of the FDH polypeptide chain, e.g., Asn136, Ala191, Tyr144, Asn234, and His263. To clarify their role in the enzyme stability, the residues were replaced with Gly by means of site-directed mutagenesis. The His263Gly mutation caused FDH destabilization and a 1.3-fold increase in the monomolecular inactivation rate constant. The replacements Ala191Gly and Asn234Gly had no significant effect on the stability. The mutations Asn136Gly and Tyr144Gly resulted in higher thermal stability and decreased the inactivation rate by 1.2- and 1.4-fold, respectively. The stabilizing effect of the Tyr144Gly mutation was shown to be additive when introduced into the previously obtained mutant FDH with enhanced thermal stability.     

Insights into the catalytic roles of the polypeptide regions in the active site of thermolysin and generation of the thermolysin variants with high activity and stability

The active site of thermolysin is composed of one zinc ion and five polypeptide regions [N-terminal sheet (Asn112-Trp115), alpha-helix 1 (Val139-Thr149), C-terminal loop 1 (Asp150-Gly162), alpha-helix 2 (Ala163-Val176) and C-terminal loop 2 (Gln225-Ser234)]. To explore their catalytic roles, we introduced single amino-acid substitutions into these regions by site-directed mutagenesis and examined their effects on the activity and stability. Seventy variants, in which one of the twelve residues (Ala113, Phe114, Trp115, Asp150, Tyr157, Gly162, Ile168, Ser169, Asp170, Asn227, Val230 and Ser234) was replaced, were produced in Escherichia coli. The hydrolytic activities of thermolysin for N-[3-(2-furyl)acryloyl]-Gly-l-Leu amide (FAGLA) and casein revealed that the N-terminal sheet and alpha-helix 2 were critical in catalysis and the C-terminal loops 1 and 2 were in substrate recognition. Twelve variants were active for both substrates. In the hydrolysis of FAGLA and N-carbobenzoxy-L-Asp-L-Phe methyl ester, the k(cat)/K(m) values of the D150E (in which Asp150 is replaced with Glu) and I168A variants were 2-3 times higher than those of the wild-type (WT) enzyme. Thermal inactivation of thermolysin at 80 degrees C was greatly suppressed with the D150H, D150W, I168A, I168H, N227A, N227H and S234A. The evidence might provide the insights into the activation and stabilization of thermolysin.     

Nucleoside diphosphate kinase from Escherichia coli.

Nucleoside diphosphate (NDP) kinase from Escherichia coli was purified to homogeneity and was crystallized. Gel filtration analysis of the purified enzyme indicated that it forms a tetramer. The enzyme was phosphorylated with [gamma-32P]ATP, and the pH stability profile of the phosphoenzyme indicated that two different amino acid residues were phosphorylated. Both a histidine residue and serine residues, including Ser-119 and Ser-121, appear to be phosphorylated. A Ser119Ala/Ser121Ala double mutant (i.e., with a Ser-to-Ala double mutation at positions 119 and 121), as well as Ser119Ala and Ser121Ala mutants, was isolated. All of these retained NDP kinase activity; also, both the Ser119Ala and Ser121Ala mutants could still be autophosphorylated. In the case of the double mutant, a slight autophosphorylation activity, which was resistant to acid treatment, was still detected, indicating that an additional minor autophosphorylation site besides His-117 exists. These results are discussed in light of the recent report of N. J. MacDonald et al. on the autophosphorylation of human NDP kinase (J. Biol. Chem. 268:25780-25789, 1993).     

Biochemical characterization of recombinant acetyl xylan esterase from Aspergillus awamori expressed in Pichia pastoris: mutational analysis of catalytic residues

We engineered an acetyl xylan esterase (AwaxeA) gene from Aspergillus awamori into a heterologous expression system in Pichia pastoris. Purified recombinant AwAXEA (rAwAXEA) displayed the greatest hydrolytic activity toward alpha-naphthylacetate (C2), lower activity toward alpha-naphthylpropionate (C3) and no detectable activity toward acyl-chain substrates containing four or more carbon atoms. Putative catalytic residues, Ser(119), Ser(146), Asp(168) and Asp(202), were substituted for alanine by site-directed mutagenesis. The biochemical properties and kinetic parameters of the four mutant enzymes were examined. The S119A and D202A mutant enzymes were catalytically inactive, whereas S146A and D168A mutants displayed significant hydrolytic activity. These observations indicate that Ser(119) and Asp(202) are important for catalysis. The S146A mutant enzyme showed lower specific activity toward the C2 substrate and higher thermal stability than wild-type enzyme. The lower activity of S146A was due to a combination of increased K(m) and decreased k(cat). The catalytic efficiency of S146A was 41% lower than that of wild-type enzyme. The synthesis of ethyl acetate was >10-fold than that of ethyl n-hexanoate synthesis for the wild-type, S146A and D168A mutant enzymes. However, the D202A showed greater synthetic activity of ethyl n-hexanoate as compared with the wild-type and other mutants.     

Utilization of an active serine 101----cysteine mutant to demonstrate the proximity of the catalytic serine 101 and histidine 237 residues in thioesterase II.

Thioesterase II is a 29-kDa monomer which, in certain specialized tissues, acts as a chain terminator in fatty acid synthesis by hydrolyzing medium-chain fatty acids from the fatty acid synthase. As with serine proteases, hydrolysis appears to involve acylation of the active site serine residue (Ser-101) assisted by a histidine, tentatively identified as His-237. To determine whether in the folded protein His-237 is close enough to accept a proton from the Ser-101 hydroxyl, we have made use of a Ser101Cys mutant which retains up to 90% of catalytic activity. Unlike the wild-type enzyme, the S101C thioesterase is inhibited with stoichiometric amounts of the bifunctional alkylating reagent 1,3-dibromopropanone. To facilitate identification of the alkylated residue(s), the keto group introduced into the dibromopropanone-modified S101C mutant was radiolabeled by reduction with sodium [3H] borohydride. The protein was then digested and the radiolabeled peptides analyzed by amino acid sequencing and mass spectrometry. The experimental data unambiguously showed that dibromopropanone cross-linked the active site Cys-101 with His-237, demonstrating that these residues are positioned within 5 A of each other. These data strongly support the hypothesis that in the wild-type thioesterase His-237 accepts a proton from Ser-101, thus increasing its nucleophilic character and improving the catalytic efficiency of the enzyme. The possibility that exchange of cysteine and serine active site residues has occurred in the evolution of thioesterases is discussed.     

Site-directed mutagenesis of the essential arginine of the formate dehydrogenase active centre

Sequence alignment shows that residue Arg 284 (according to the numbering of the residues in formate dehydrogenase, FDH, from the methylotrophic bacterium Pseudomonas sp. 101) is conserved in NAD-dependent FDHs and D-specific 2-hydroxyacid dehydrogenases. Mutation of Arg 284 to glutamine and alanine results in a change of the catalytic, thermodynamic and spectral properties of FDH. In comparison to wild-type, the affinity of the mutants for the substrate (K(formate)m) or the transition state analogue (K(azide)i) decreases and correlates with the ability of the side chain of residue 284 to form H-bonds. In contrast, the affinity for the coenzyme (K(NAD)d or K(NAD)m) is either not affected or increases and correlates inversely with the partial positive charge of the side chain. The temperature dependence of circular dichroism (CD) spectra of the wild-type FDH and its Ala mutant has been studied over the 5-90 degrees C temperature range. Both proteins reveal regions of enhanced conformational mobility at the predenaturing temperatures (40-55 degrees C) associated with a change of enzyme kinetic parameters and a co-operative transition around 55-70 degrees C which is followed by the loss of enzyme activity. CD spectra of the wild-type and mutant proteins were deconvoluted and contributions from various types of secondary structure estimated. It is shown that the co-operative transition at 55-70 degrees C in the FDH protein globule is triggered by a loss of alpha-helical secondary structure. The results confirm the conclusion, from the crystal structures, that Arg 284 is directly involved in substrate binding. In addition this residue seems to exert a major structural role by supporting the catalytic conformation of the enzyme active centre.     

Improving the affinity and activity of CYP101D2 for hydrophobic substrates

CYP101D2 is a cytochrome P450 monooxygenase from Novosphingobium aromaticivorans which is closely related to CYP101A1 (P450cam) from Pseudomonas putida. Both enzymes selectively hydroxylate camphor to 5-exo-hydroxycamphor, and the residues that line the active sites of both enzymes are similar including the pre-eminent Tyr96 residue. However, Met98 and Leu253 in CYP101D2 replace Phe98 and Val247 in CYP101A1, and camphor binding only results in a maximal change in the spin state to 40 % high-spin. Substitutions at Tyr96, Met98 and Leu253 in CYP101D2 reduced both the spin state shift on camphor binding and the camphor oxidation activity. The Tyr96Ala mutant increased the affinity of CYP101D2 for hydrocarbon substrates including adamantane, cyclooctane, hexane and 2-methylpentane. The monooxygenase activity of the Tyr96Ala variant towards alkane substrates was also enhanced compared with the wild-type enzyme. The crystal structure of the substrate-free form of this variant shows the enzyme in an open conformation (PDB: 4DXY), similar to that observed with the wild-type enzyme (PDB: 3NV5), with the side chain of Ala96 pointing away from the heme. Despite this, the binding and activity data suggest that this residue plays an important role in substrate binding, evidencing that the enzyme probably undergoes catalysis in a more closed conformation, similar to those observed in the crystal structures of CYP101A1 (PDB: 2CPP) and CYP101D1 (PDB: 3LXI).     

Stabilization of NAD-dependent formate dehydrogenase from Candida boidinii by site-directed mutagenesis of cysteine residues.

The gene of the NAD-dependent formate dehydrogenase (FDH) from the yeast Candida boidinii was cloned by PCR using genomic DNA as a template. Expression of the gene in Escherichia coli yielded functional FDH with about 20% of the soluble cell protein. To confirm the hypothesis of a thiol-coupled inactivation process, both cysteine residues in the primary structure of the enzyme have been exchanged by site-directed mutagenesis using a homology model based on the 3D structure of FDH from Pseudomonas sp. 101 and from related dehydrogenases. Compared to the wt enzyme, most of the mutants were significantly more stable towards oxidative stress in the presence of Cu(II) ions, whereas the temperature optima and kinetic constants of the enzymatic reaction are not significantly altered by the mutations. Determination of the Tm values revealed that the stability at temperatures above 50 degrees C is optimal for the native and the recombinant wt enzyme (Tm 57 degrees C), whereas the Tm values of the mutant enzymes vary in the range 44-52 degrees C. Best results in initial tests concerning the application of the enzyme for regeneration of NADH in biotransformation of trimethyl pyruvate to Ltert leucine were obtained with two mutants, FDHC23S and FDHC23S/C262A, which are significantly more stable than the wt enzyme.     

The allosteric activator ATP induces a substrate-dependent alteration of the quaternary structure of a mutant aspartate transcarbamoylase impaired in active site closure.

Aspartate transcarbamoylase from Escherichia coli shows homotropic cooperativity for aspartate as well as heterotropic regulation by nucleotides. Structurally, it consists of two trimeric catalytic subunits and three dimeric regulatory subunits, each chain being comprised of two domains. Glu-50 and Ser-171 are involved in stabilizing the closed conformation of the catalytic chain. Replacement of Glu-50 or Ser-171 by Ala in the holoenzyme has been shown previously to result in marked decreases in the maximal observed specific activity, homotropic cooperativity, and affinity for aspartate (Dembowski NJ, Newton CJ, Kantrowitz ER, 1990, Biochemistry 29:3716-3723; Newton CJ, Kantrowitz ER, 1990, Biochemistry 29:1444-1451). We have constructed a double mutant enzyme combining both mutations. The resulting Glu-50/ser-171-->Ala enzyme is 9-fold less active than the Ser-171-->Ala enzyme, 69-fold less active than the Glu-50-->Ala enzyme, and shows 1.3-fold and 1.6-fold increases in the [S]0.5Asp as compared to the Ser-171-->Ala and Glu-50-->Ala enzymes, respectively. However, the double mutant enzyme exhibits some enhancement of homotropic cooperativity with respect to aspartate, relative to the single mutant enzymes. At subsaturating concentrations of aspartate, the Glu-50/Ser-171 -->Ala enzyme is activated less by ATP than either the Glu-50-->Ala or Ser-171-->Ala enzyme, whereas CTP inhibition is intermediate between that of the two single mutants. As opposed to the wild-type enzyme, the Glu-50/Ser-171 -->Ala enzyme is activated by ATP and inhibited by CTP at saturating concentrations of aspartate. Structural analysis of the Ser-171-->Ala and Glu-50/Ser-171-->Ala enzymes by solution X-ray scattering indicates that both mutants exist in the same T quaternary structure as the wild-type enzyme in the absence of ligands, and in the same R quaternary structure in the presence of saturating N-(phosphonoacetyl)-L-aspartate. However, saturating concentrations of carbamoyl phosphate and succinate are unable to convert a significant fraction of either mutant enzyme population to the R quaternary structure, as has been observed previously for the Glu-50-->Ala enzyme. The curves for both the Ser-171-->Ala and Glu-50/Ser-171-->Ala enzymes obtained in the presence of substoichiometric amounts of PALA are linear combinations of the two extreme T and R states. The structural consequences of nucleotide binding to these two enzymes were also investigated. Most surprisingly, the direction and amplitude of the effect of ATP upon the double mutant enzyme were shown to vary depending upon the substrate analogue used.     

Mutational analysis of feedback inhibition and catalytic sites of prephenate dehydratase from<Emphasis Type="Italic"> Corynebacterium glutamicum</Emphasis>

Prephenate dehydratase is a key regulatory enzyme in the phenylalanine-specific pathway of Corynebacterium glutamicum. PCR-based random mutagenesis and functional complementation were used to screen for m-fluorophenylalanine (mFP)-resistant mutants. Comparison of the amino acid sequence of the mutant prephenate dehydratases indicated that Ser-99 plays a role in the feedback regulation of the enzyme. When Ser-99 of the wild-type enzyme was replaced by Met, the specific activity of the mutant enzyme was 30% lower than that of the wild-type. The Ser99Met mutant was active in the presence of 50 M phenylalanine, whereas the wild-type enzyme was not. The functional roles of the eight conserved residues of prephenate dehydratase were investigated by site-directed mutagenesis. Glu64Asp substitution reduced enzyme activity by 15%, with a 4.5- and 1.7-fold increase in K _m and k _cat values, respectively. Replacement of Thr-183 by either Ala or Tyr resulted in a complete loss of enzyme activity. Substitution of Arg-184 with Leu resulted in a 50% decrease of enzyme activity. The specific activity for Phe185Tyr was more than 96% lower than that of the wild-type, and the K _m value was 26-fold higher. Alterations in the conserved Asp-76, Glu-89, His-115, and Arg-236 residues did not cause a significant change in the K _m and k _cat values. These results indicated that Glu-64, Thr-183, Arg-184, and Phe-185 residues might be involved in substrate binding and/or catalytic activity.     

Amino acid residues conferring herbicide tolerance in tobacco acetolactate synthase

Acetolactate synthase (ALS) is the common enzyme in the biosynthetic pathways leading to valine, leucine, and isoleucine in plants and microorganisms. ALS is the target site of several classes of structurally unrelated herbicides including sulfonylureas, imidazolinones, and triazolopyrimidines. To identify the residues conferring herbicide tolerance in tobacco ALS, site-directed mutagenesis for three residues, Ala121, Pro187 and Ser652, was performed. Mutant A121T showed strong resistance to Londax (a sulfonylurea) and Cadre (an imidazolinone), while mutant S652T was resistant only to Cadre. The S652N mutation abolished the binding affinity of FAD, and inactivated the enzyme. Double mutation of Ala121 and Ser652 with Thr yielded a mutant highly tolerant to Londax, Cadre, and TP (a triazolopyrimidine sulfonamide), but has enzymatic properties similar to those of wild-type. Substitution of Pro187 with Ser resulted in the enzyme highly susceptible to oxidation and fragmentation. These results suggest that two residues Ala121 and Ser652 are potent residues conferring herbicide resistance in tobacco ALS, and that double mutation of Ala121 and Ser652 by Thr can confer stronger tolerance to Londax, Cadre, and TP.     

Phosphorylation of mammalian eukaryotic translation initiation factor 6 and its Saccharomyces cerevisiae homologue Tif6p: evidence that phosphorylation of Tif6p regulates its nucleocytoplasmic distribution and is required for yeast cell growth

The synthesis of 60S ribosomal subunits in Saccharomyces cerevisiae requires Tif6p, the yeast homologue of mammalian eukaryotic translation initiation factor 6 (eIF6). In the present work, we have isolated a protein kinase from rabbit reticulocyte lysates on the basis of its ability to phosphorylate recombinant human eIF6. Mass spectrometric analysis as well as antigenic properties of the purified kinase identified it as casein kinase I. The site of in vitro phosphorylation, which is highly conserved from yeast to mammals, was identified as the serine residues at positions 174 (major site) and 175 (minor site). The homologous yeast protein Tif6p was also phosphorylated in vivo in yeast cells. Mutation of Tif6p at serine-174 to alanine reduced phosphorylation drastically and caused loss of cell growth and viability. When both Ser-174 and Ser-175 were mutated to alanine, phosphorylation of Tif6p was completely abolished. Furthermore, while wild-type Tif6p was distributed both in nuclei and the cytoplasm of yeast cells, the mutant Tif6p (with Ser174Ala and Ser175Ala) became a constitutively nuclear protein. These results suggest that phosphorylatable Ser-174 and Ser-175 play a critical role in the nuclear export of Tif6p.     

Structural and kinetic studies on the Ser101Ala variant of choline oxidase: Catalysis by compromise

The oxidation of choline catalyzed by choline oxidase includes two reductive half-reactions where FAD is reduced by the alcohol substrate and by an aldehyde intermediate transiently formed in the reaction. Each reductive half-reaction is followed by an oxidative half-reaction where the reduced flavin is oxidized by oxygen. Here, we have used mutagenesis to prepare the Ser101Ala mutant of choline oxidase and have investigated the impact of this mutation on the structural and kinetic properties of the enzyme. The crystallographic structure of the Ser101Ala enzyme indicates that the only differences between the mutant and wild-type enzymes are the lack of a hydroxyl group on residue 101 and a more planar configuration of the flavin in the mutant enzyme. Kinetics established that replacement of Ser101 with alanine yields a mutant enzyme with increased efficiencies in the oxidative half-reactions and decreased efficiencies in the reductive half-reactions. This is accompanied by a significant decrease in the overall rate of turnover with choline. Thus, this mutation has revealed the importance of a specific residue for the optimization of the overall turnover of choline oxidase, which requires fine-tuning of four consecutive half-reactions for the conversion of an alcohol to a carboxylic acid.     

Ser515 phosphorylation-independent regulation of cytosolic phospholipase A2alpha (cPLA2alpha) by calmodulin-dependent protein kinase: possible interaction with catalytic domain A of cPLA2alpha

Calmodulin (CaM)-dependent protein kinase (CaM kinase) is proposed to regulate the type alpha of cytosolic phospholipase A(2) (cPLA(2)alpha), which has a dominant role in the release of arachidonic acid (AA), via phosphorylation of Ser515 of the enzyme. However, the exact role of CaM kinase in the activation of cPLA(2)alpha has not been well established. We investigated the effects induced by transfection with mutant cPLA(2)alpha and inhibitors for CaM and CaM kinase on the Ca(2+)-stimulated release of AA and translocation of cPLA(2)alpha. The mutation of Ser515 to Ala (S515A) did not change cPLA(2)alpha activity, although S228A and S505A completely and partially decreased the activity, respectively. Stimulation with hydrogen peroxide (H(2)O(2), 1 mM) and A23187 (10 microM) markedly released AA in C12 cells expressing S515A and wild-type cPLA(2)alpha, but the responses in C12-S505A, C12-S727A, and C12-S505A/S515A/S727A (AAA) cells were reduced. In HEK293T cells expressing cPLA(2)alpha, A23187 caused the translocation of the wild-type, the every mutants, cPLA(2)alpha-C2 domain, and cPLA(2)alpha-Delta397-749 lacking proposed phosphorylation sites such as Ser505 and Ser515. Treatment with inhibitors of CaM (W-7) and CaM kinase (KN-93) at 10 microM significantly decreased the release of AA in C12-cPLA(2)alpha cells and C12-S515A cells. KN-93 inhibited the A23187-induced translocation of the wild-type, S515A, AAA and cPLA(2)alpha-Delta397-749, but not cPLA(2)alpha-C2 domain. Our findings show a possible effect of CaM kinase on cPLA(2)alpha in a catalytic domain A-dependent and Ser515-independent manner.     

Stabilization of plant formate dehydrogenase by rational design

Recombinant formate dehydrogenase (FDH, EC 1.2.1.2) from soy Glycine max (SoyFDH) has the lowest values of Michaelis constants for formate and NAD⁺ among all studied formate dehydrogenases from different sources. Nevertheless, it also has the lower thermal stability compared to enzymes from bacteria and yeasts. The alignment of full sequences of FDHs from different sources as well as structure of apo- and holo-forms of SoyFDH has been analyzed. Ten mutant forms of SoyFDH were obtained by site-directed mutagenesis. All of them were purified to homogeneity and their thermal stability and substrate specificity were studied. Thermal stability was investigated by studying the inactivation kinetics at different temperatures and by differential scanning calorimetry (DSC). As a result, single-point (Ala267Met) and double mutants (Ala267Met/Ile272Val) were found to be more stable than the wild-type enzyme at high temperatures. The stabilization effect depends on temperature, and at 52°C it was 3.6- and 11-fold, respectively. These mutants also showed higher melting temperatures in DSC experiments — the differences in maxima of the melting curves (T _m) for the single and double mutants were 2.7 and 4.6°C, respectively. For mutations Leu24Asp and Val127Arg, the thermal stability at 52°C decreased 5- and 2.5-fold, respectively, and the T _m decreased by 3.5 and 1.7°C, respectively. There were no differences in thermal stability of six mutant forms of SoyFDH — Gly18Ala, Lys23Thr, Lys109Pro, Asn247Glu, Val281Ile, and Ser354Pro. Analysis of kinetic data showed that for the enzymes with mutations Val127Arg and Ala267Met the catalytic efficiency increased 1.7- and 2.3-fold, respectively.     

Serine 132 is the C3 covalent attachment point on the CH1 domain of human IgG1.

The covalent binding of C3 (complement component C3) to antigen-antibody complexes (Ag.Ab; immune complexes (ICs)) is a key event in the uptake, transport, presentation, and elimination of Ag in the form of Ag.Ab.C3b (IC.C3b). Upon interaction of C3 with IgG.IC, C3b.C3b.IgG covalent complexes are formed that are detected on SDS-polyacrylamide gel electrophoresis by two bands corresponding to C3b.C3b (band A) and C3b.IgG (band B) covalent complexes. This allows one to evaluate the covalent binding of C3b to IgG antibodies. It has been described that C3b can attach to both the Fab (on the CH1 domain) and the Fc regions of IgG. Here the covalent interaction of C3b to the CH1 domain, a region previously described spanning residues 125-147, has been studied. This region of the CH1 domain is exposed to solvent and contains a cluster of six potential acceptor sites for ester bond formation with C3b (four Ser and two Thr). A set of 10 mutant Abs were generated with the putative acceptor residues substituted by Ala, and we studied their covalent interaction with C3b. Single (Ser-131, Ser-132, Ser-134, Thr-135, Ser-136, and Thr-139), double (positions 131-132), and multiple (positions 134-135-136, 131-132-134-135-136, and 131-132-134-135-136-139) mutants were produced. None of the mutants (single, double, or multiple) abolished completely the ability of IgG to bind C3b, indicating the presence of C3b binding regions other than in the CH1 domain. However, all mutant Abs, in which serine at position 132 was replaced by Ala, showed a significant decrease in the ability to form C3b.IgG covalent complexes, whereas the remaining mutants had normal activity. In addition we examined ICs using the F(ab')2 fragment of the mutant Abs, and only those containing Ala at position 132 (instead of Ser) failed to bind C3b. Thus Ser-132 is the binding site for C3b on the CH1 domain of the heavy chain, in the Fab region of human IgG.     

Crystal structures of aconitase with isocitrate and nitroisocitrate bound.

The crystal structures of mitochondrial aconitase with isocitrate and nitroisocitrate bound have been solved and refined to R factors of 0.179 and 0.161, respectively, for all observed data in the range 8.0-2.1 A. Porcine heart enzyme was used for determining the structure with isocitrate bound. The presence of isocitrate in the crystals was corroborated by M?ssbauer spectroscopy. Bovine heart enzyme was used for determining the structure with the reaction intermediate analogue nitroisocitrate bound. The inhibitor binds to the enzyme in a manner virtually identical to that of isocitrate. Both compounds bind to the unique Fe atom of the [4Fe-4S] cluster via a hydroxyl oxygen and one carboxyl oxygen. A H2O molecule is also bound, making Fe six-coordinate. The unique Fe is pulled away approximately 0.2 A from the corner of the cubane compared to the position it would occupy in a symmetrically ligated [4Fe-4S] cluster. At least 23 residues from all four domains of aconitase contribute to the active site. These residues participate in substrate recognition (Arg447, Arg452, Arg580, Arg644, Gln72, Ser166, Ser643), cluster ligation and interaction (Cys358, Cys421, Cys424, Asn258, Asn446), and hydrogen bonds supporting active site side chains (Ala74, Asp568, Ser571, Thr567). Residues implicated in catalysis are Ser642 and three histidine-carboxylate pairs (Asp100-His101, Asp165-His147, Glu262-His167). The base necessary for proton abstraction from C beta of isocitrate appears to be Ser642; the O gamma atom is proximal to the calculated hydrogen position, while the environment of O gamma suggests stabilization of an alkoxide (an oxyanion hole formed by the amide and side chain of Arg644). The histidine-carboxylate pairs appear to be required for proton transfer reactions involving two oxygens bound to Fe, one derived from solvent (bound H2O) and one derived from substrate hydroxyl. Each oxygen is in contact with a histidine, and both are in contact with the side chain of Asp165, which bridges the two sites on the six-coordinate Fe.