Crystal structure of the reduced Schiff-base intermediate complex of transaldolase B from Escherichia coli: mechanistic implications for class I aldolases.

Transaldolase catalyzes transfer of a dihydroxyacetone moiety from a ketose donor to an aldose acceptor. During catalysis, a Schiff-base intermediate between dihydroxyacetone and the epsilon-amino group of a lysine residue at the active site of the enzyme is formed. This Schiff-base intermediate has been trapped by reduction with potassium borohydride, and the crystal structure of this complex has been determined at 2.2 A resolution. The overall structures of the complex and the native enzyme are very similar; formation of the intermediate induces no large conformational changes. The dihydroxyacetone moiety is covalently linked to the side chain of Lys 132 at the active site of the enzyme. The Cl hydroxyl group of the dihydroxyacetone moiety forms hydrogen bonds to the side chains of residues Asn 154 and Ser 176. The C3 hydroxyl group interacts with the side chain of Asp 17 and Asn 35. Based on the crystal structure of this complex a reaction mechanism for transaldolase is proposed.     

Lysine 335, part of the KMSKS signature sequence, plays a crucial role in the amino acid activation catalysed by the methionyl-tRNA synthetase from Escherichia coli.

The KMSKS pattern, conserved among several aminoacyl-tRNA synthetase sequences, was first recognized in the Escherichia coli methionyl-tRNA synthetase through affinity labelling with an oxidized reactive derivative of tRNA(Met)f. Upon complex formation, two lysine residues of the methionyl-tRNA synthetase (Lys61 and 335, the latter being part of the KMSKS sequence) could be crosslinked by the 3'-acceptor end of the oxidized tRNA. Identification of an equivalent reactive lysine residue at the active centre of tyrosyl-tRNA synthetase designated the KMSKS sequence as a putative component of the active site of methionyl-tRNA synthetase. To probe the functional role of the labelled lysine residue within the KMSKS pattern, two variants of methionyl-tRNA synthetase containing a glutamine residue at either position 61 or 335 were constructed by using site-directed mutagenesis. Substitution of Lys61 slightly affected the enzyme activity. In contrast, the enzyme activities were very sensitive to the substitution of Lys335 by Gln. Pre-steady-state analysis of methionyladenylate synthesis demonstrated that this substitution rendered the enzyme unable to stabilize the transition state complex in the methionine activation reaction. A similar effect was obtained upon substituting Lys335 by an alanine instead of a glutamine residue, thereby excluding an effect specific for the glutamine side-chain. Furthermore, the importance of the basic character of Lys335 was investigated by studying mutants with a glutamate or an arginine residue at this position. It is concluded that the N-6-amino group of Lys335 plays a crucial role in the activation of methionine, mainly by stabilizing the transient complex on the way to methionyladenylate, through interaction with the pyrophosphate moiety of bound ATP-Mg2+. We propose, therefore, that the KMSKS pattern in the structure of an aminoacyl-tRNA synthetase sequence represents a signature sequence characteristic of both the pyrophosphate subsite and the catalytic centre.     

Experimental and computational investigations of Ser10 and Lys13 in the binding and cleavage of DNA substrates by Escherichia coli DNA topoisomerase I

Ser10 and Lys13 found near the active site tyrosine of Escherichia coli DNA topoisomerase I are conserved among the type IA topoisomerases. Site-directed mutagenesis of these two residues to Ala reduced the relaxation and DNA cleavage activity, with a more severe effect from the Lys13 mutation. Changing Ser10 to Thr or Lys13 to Arg also resulted in loss of DNA cleavage and relaxation activity of the enzyme. In simulations of the open form of the topoisomerase–DNA complex, Lys13 interacts directly with Glu9 (proposed to be important in the catalytic mechanism). This interaction is removed in the K13A mutant, suggesting the importance of lysine as either a proton donor or a stabilizing cation during strand cleavage, while the Lys to Arg mutation significantly distorts catalytic residues. Ser10 forms a direct hydrogen bond with a phosphate group near the active site and is involved in direct binding of the DNA substrate; this interaction is disturbed in the S10A and S10T mutants. This combination of a lysine and a serine residue conserved in the active site of type IA topoisomerases may be required for correct positioning of the scissile phosphate and coordination of catalytic residues relative to each other so that DNA cleavage and subsequent strand passage can take place.     

Structure of the conserved core of the yeast Dot1p, a nucleosomal histone H3 lysine 79 methyltransferase

Methylation of Lys79 on histone H3 by Dot1p is important for gene silencing. The elongated structure of the conserved core of yeast Dot1p contains an N-terminal helical domain and a seven-stranded catalytic domain that harbors the binding site for the methyl-donor and an active site pocket sided with conserved hydrophobic residues. The S-adenosyl-L-homocysteine exhibits an extended conformation distinct from the folded conformation observed in structures of SET domain histone lysine methyltransferases. A catalytic asparagine (Asn479), located at the bottom of the active site pocket, suggests a mechanism similar to that employed for amino methylation in DNA and protein glutamine methylation. The acidic, concave cleft between the two domains contains two basic residue binding pockets that could accommodate the outwardly protruding basic side chains around Lys79 of histone H3 on the disk-like nucleosome surface. Biochemical studies suggest that recombinant Dot1 proteins are active on recombinant nucleosomes, free of any modifications.     

Refined crystal structure of beta-lactamase from Staphylococcus aureus PC1 at 2.0 A resolution

The crystal structure of a class A beta-lactamase from Staphylococcus aureus PC1 has been refined at 2.0 A resolution. The resulting crystallographic R-factor (R = sigma h parallel Fo[-]Fc parallel/sigma h[Fo], where [Fo] and [Fc] are the observed and calculated structure factor amplitudes, respectively), is 0.163 for the 17,547 reflections with I greater than or equal to 2 sigma (I) within the 8.0 A to 2.0 A resolution range. The molecule consists of two closely associated domains. One domain is formed by a five-stranded antiparallel beta-sheet with three helices packing against a face of the sheet. The second domain is formed mostly by helices that pack against the second face of the sheet. The active site is located in the interface between the two domains, and many of the residues that form it are conserved in all known sequences of class A beta-lactamases. Similar to the serine proteases, an oxyanion hole is implicated in catalysis. It is formed by two main-chain nitrogen atoms, that of the catalytic seryl residue, Ser70, and that of Gln237 on an edge beta-strand of the major beta-sheet. Ser70 is interacting with another conserved seryl residue, Ser130, located between the two ammonium groups of the functionally important lysine residues, Lys73 and Lys234. Such intricate interactions point to a possible catalytic role for this second seryl residue. Another key catalytic residue is Glu166. There are several unusual structural features associated with the active site. (1) A cis peptide bond has been identified between the catalytic Glu166 and Ile167. (2) Ala69 and Leu220 have strained phi, psi dihedral angles making close contacts that restrict the conformation of the active site beta-strand involved in the formation of the oxyanion hole. (3) A buried aspartate residue, the conserved Asp233, is located next to the active site Lys234. It is interacting with another buried aspartyl residue, Asp246. An internal solvent molecule is also involved, but the rest of its interactions with the protein indicate it is not a cation. (4) Another conserved aspartyl residue that is desolvated is Asp131, adjacent to Ser130. Its charge is stabilized by interactions with four main-chain nitrogen atoms. (5) An internal cavity underneath the active site depression is filled with six solvent molecules. This, and an adjacent cavity occupied by three solvent molecules partially separate the omega-loop associated with the active site from the rest of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cassette mutagenesis of lysine 130 of human glutamate dehydrogenase. An essential residue in catalysis.

It has been suggested that reactive lysine residue(s) may play an important role in the catalytic activities of glutamate dehydrogenase (GDH). There are, however, conflicting views as to whether the lysine residues are involved in Schiff's base formation with catalytic intermediates, stabilization of negatively charged groups or the carbonyl group of 2-oxoglutarate during catalysis, or some other function. We have expanded on these speculations by constructing a series of cassette mutations at Lys130, a residue that has been speculated to be responsible for the activity of GDH and the inactivation of GDH by pyridoxal 5'-phosphate (PLP). For these studies, a 1557-bp gene that encodes human GDH has been synthesized and inserted into Escherichia coli expression vectors. The mutant enzymes containing Glu, Gly, Met, Ser, or Tyr at position 130, as well as the wild-type human GDH encoded by the synthetic gene, were efficiently expressed as a soluble protein and are indistinguishable from that isolated from human and bovine tissues. Despite an approximately 400-fold decrease in the respective apparent Vmax of the Lys130 mutant enzymes, apparent Km values for NADH and 2-oxoglutarate were almost unchanged, suggesting the direct involvement of Lys130 in catalysis rather than in the binding of coenzyme or substrate. Unlike the wild-type GDH, the mutant enzymes were unable to interact with PLP, indicating that Lys130 plays an important role in PLP binding. The results with analogs of PLP suggest that the aldehyde moiety of PLP, but not the phosphate moiety, is required for efficient binding to GDH.     

Leishmania mexicana glycerol-3-phosphate dehydrogenase showed conformational changes upon binding a bi-substrate adduct

Certain pathogenic trypanosomatids are highly dependent on glycolysis for ATP production, and hence their glycolytic enzymes, including glycerol-3-phosphate dehydrogenase (GPDH), are considered attractive drug targets. The ternary complex structure of Leishmania mexicana GPDH (LmGPDH) with dihydroxyacetone phosphate (DHAP) and NAD(+) was determined to 1.9A resolution as a further step towards understanding this enzyme's mode of action. When compared with the apo and binary complex structures, the ternary complex structure shows an 11 degrees hinge-bending motion of the C-terminal domain with respect to the N-terminal domain. In addition, residues in the C-terminal domain involved in catalysis or substrates binding show significant movements and a previously invisible five-residue loop region becomes well ordered and participates in NAD(+) binding. Unexpectedly, DHAP and NAD(+) appear to form a covalent bond, producing an adduct in the active site of LmGPDH. Modeling a ternary complex glycerol 3-phosphate (G3P) and NAD(+) with LmGPDH identified ten active site residues that are highly conserved among all GPDHs. Two lysine residues, Lys125 and Lys210, that are presumed to be critical in catalysis, were mutated resulting in greatly reduced catalytic activity. Comparison with other structurally related enzymes found by the program DALI suggested Lys210 as a key catalytic residue, which is located on a structurally conserved alpha-helix. From the results of site-directed mutagenesis, molecular modeling and comparison with related dehydrogenases, a catalytic mechanism of LmGPDH and a possible evolutionary scenario of this group of dehydrogenases are proposed.     

Analysis of the class I aldolase binding site architecture based on the crystal structure of 2-deoxyribose-5-phosphate aldolase at 0.99A resolution

The crystal structure of the bacterial (Escherichia coli) class I 2-deoxyribose-5-phosphate aldolase (DERA) has been determined by Se-Met multiple anomalous dispersion (MAD) methods at 0.99A resolution. This structure represents the highest-resolution X-ray structure of an aldolase determined to date and enables a true atomic view of the enzyme. The crystal structure shows the ubiquitous TIM alpha/beta barrel fold. The enzyme contains two lysine residues in the active site. Lys167 forms the Schiff base intermediate, whereas Lys201, which is in close vicinity to the reactive lysine residue, is responsible for the perturbed pK(a) of Lys167 and, hence, also a key residue in the reaction mechanism. DERA is the only known aldolase that is able to use aldehydes as both aldol donor and acceptor molecules in the aldol reaction and is, therefore, of particular interest as a biocatalyst in synthetic organic chemistry. The uncomplexed DERA structure enables a detailed comparison with the substrate complexes and highlights a conformational change in the phosphate-binding site. Knowledge of the enzyme active-site environment has been the basis for exploration of catalysis of non-natural substrates and of mutagenesis of the phosphate-binding site to expand substrate specificity. Detailed comparison with other class I aldolase enzymes and DERA enzymes from different organisms reveals a similar geometric arrangement of key residues and implies a potential role for water as a general base in the catalytic mechanism.     

Widely distributed residues in thymosin beta4 are critical for actin binding

We have investigated the contributions of hydrophobic residues, the conserved and variable proline residues, and the conserved lysine residues to the affinity and kinetics of thymosin beta4 (Tbeta4) binding to MgATP-actin monomers. Pro4, Lys18, Lys19, Pro27, Leu28, Pro29, and Ile34 were substituted with alanine residues. Mutagenesis of Pro4 or Pro27 has little effect (or=10-fold, but the kinetic basis of the lower stability varies among the mutants. Substitution of the conserved lysine residues weakens the affinity by slowing association and accelerating dissociation. Substitution of hydrophobic residue Leu28 or Ile34 weakens the affinity by accelerating dissociation. These results favor a reaction mechanism in which Tbeta4 binds actin monomers following a two-step mechanism in which the formation of a bimolecular complex is followed by isomerization to a strong binding state that is coupled to the formation of widely distributed hydrophobic contacts. The isomerization equilibrium is slowed by mutagenesis of Pro29, as revealed by the double-exponential time course of association. Mutagenesis of Pro4 or Pro27 accelerates binding and dissociation but minimally affects the binding affinity (相似文献   

Acetylation of estrogen receptor alpha by p300 at lysines 266 and 268 enhances the deoxyribonucleic acid binding and transactivation activities of the receptor

Using a variety of biochemical and cell-based approaches, we show that estrogen receptor alpha (ERalpha) is acetylated by the p300 acetylase in a ligand- and steroid receptor coactivator-dependent manner. Using mutagenesis and mass spectrometry, we identified two conserved lysine residues in ERalpha (Lys266 and Lys268) that are the primary targets of p300-mediated acetylation. These residues are acetylated in cells, as determined by immunoprecipitation-Western blotting experiments using an antibody that specifically recognizes ERalpha acetylated at Lys266 and Lys268. The acetylation of ERalpha by p300 is reversed by native cellular deacetylases, including trichostatin A-sensitive enzymes (i.e. class I and II deacetylases) and nicotinamide adenine dinucleotide-dependent/nicotinamide-sensitive enzymes (i.e. class III deacetylases, such as sirtuin 1). Acetylation at Lys266 and Lys268, or substitution of the same residues with glutamine (i.e. K266/268Q), a residue that mimics acetylated lysine, enhances the DNA binding activity of ERalpha in EMSAs. Likewise, substitution of Lys266 and Lys268 with glutamine enhances the ligand-dependent activity of ERalpha in a cell-based reporter gene assay. Collectively, our results implicate acetylation as a modulator of the ligand-dependent gene regulatory activity of ERalpha. Such regulation is likely to play a role in estrogen-dependent signaling outcomes in a variety of estrogen target tissues in both normal and pathological states.     

The catalytic mechanism of indole-3-glycerol phosphate synthase: crystal structures of complexes of the enzyme from Sulfolobus solfataricus with substrate analogue,substrate, and product

Indoleglycerol phosphate synthase catalyzes the ring closure of an N-alkylated anthranilate to a 3-alkyl indole derivative, a reaction requiring Lewis acid catalysis in vitro. Here, we investigated the enzymatic reaction mechanism through X-ray crystallography of complexes of the hyperthermostable enzyme from Sulfolobus solfataricus with the substrate 1-(o-carboxyphenylamino) 1-deoxyribulose 5-phosphate, a substrate analogue and the product indole-3-glycerol phosphate. The substrate and the substrate analogue are bound to the active site in a similar, extended conformation between the previously identified phosphate binding site and a hydrophobic pocket for the anthranilate moiety. This binding mode is unproductive, because the carbon atoms that are to be joined are too far apart. The indole ring of the bound product resides in a second hydrophobic pocket adjacent to that of the anthranilate moiety of the substrate. Although the hydrophobic moiety of the substrate moves during catalysis from one hydrophobic pocket to the other, the triosephosphate moiety remains rigidly bound to the same set of hydrogen-bonding residues. Simultaneously, the catalytically important residues Lys53, Lys110 and Glu159 maintain favourable distances to the atoms of the ligand undergoing covalent changes. On the basis of these data, the structures of two putative catalytic intermediates were modelled into the active site. This new structural information and the modelling studies provide further insight into the mechanism of enzyme-catalyzed indole synthesis. The charged epsilon-amino group of Lys110 is the general acid, and the carboxylate group of Glu159 is the general base. Lys53 guides the substrate undergoing conformational transitions during catalysis, by forming a salt-bridge to the carboxylate group of its anthranilate moiety.     

Lys300 plays a major role in the catalytic mechanism of maize polyamine oxidase

Maize polyamine oxidase (MPAO) is a flavin adenine dinucleotide (FAD)-dependent enzyme that catalyses the oxidation of spermine and spermidine at the secondary amino groups. The structure of MPAO indicates a 30-A long U-shaped tunnel that forms the catalytic site, with residues Glu62 and Glu170 located close to the enzyme-bound FAD and residue Tyr298 in close proximity to Lys300, which in turn is hydrogen-bonded to the flavin N(5) atom via a water molecule (HOH309). To provide insight into the role of these residues in the catalytic mechanism of FAD reduction, we have performed steady-state and stopped-flow studies with wild-type, Glu62Gln, Glu170Gln, Tyr298Phe, and Lys300Met MPAO enzymes. We show that the steady-state enzyme activity is governed by an ionisable group with a macroscopic pK(a) of approximately 5.8. Kinetic analysis of the Glu62Gln, Glu170Gln, and Tyr298Phe MPAO enzymes have indicated (i) only small perturbations in catalytic activity as a result of mutation and (ii) steady-state pH profiles essentially unaltered when compared to the wild-type enzyme, suggesting that these residues do not play a critical role in the reaction mechanism. These kinetic observations are consistent with computational calculations that suggest that Glu62 and Glu170 are protonated over the pH range accessible to kinetic studies. Substitution of Lys300 with Met in MPAO resulted in a 1400-fold decrease in the rate of flavin reduction and a 160-fold decrease in the equilibrium dissociation constant for the Lys300Met-spermidine complex, consistent with a major role for this residue in the mechanism of substrate oxidation. A sizable solvent isotope effect (SIE = 5) accompanies FAD reduction in the wild-type enzyme and steady-state turnover (SIE = 2.3) of MPAO, consistent with the reductive half-reaction of MPAO making a major contribution to rate limitation in steady-state turnover. Studies using the enzyme-monitored turnover method indicate that oxidized FAD is the prominent form during steady-state turnover, consistent with the reductive half-reaction being rate-limiting. Our studies indicate the importance of Lys300 and probable importance of HOH309 to the mechanism of flavin reduction in MPAO. Possible roles for Lys300 and water in the mechanism of flavin reduction are discussed.     

DNA stabilization at the Bacillus subtilis PolX core��a binding model to coordinate polymerase, AP-endonuclease and 3��-5�� exonuclease activities

Family X DNA polymerases (PolXs) are involved in DNA repair. Their binding to gapped DNAs relies on two conserved helix-hairpin-helix motifs, one located at the 8-kDa domain and the other at the fingers subdomain. Bacterial/archaeal PolXs have a specifically conserved third helix-hairpin-helix motif (GFGxK) at the fingers subdomain whose putative role in DNA binding had not been established. Here, mutagenesis at the corresponding residues of Bacillus subtilis PolX (PolXBs), Gly130, Gly132 and Lys134 produced enzymes with altered DNA binding properties affecting the three enzymatic activities of the protein: polymerization, located at the PolX core, 3′-5′ exonucleolysis and apurinic/apyrimidinic (AP)-endonucleolysis, placed at the so-called polymerase and histidinol phosphatase domain. Furthermore, we have changed Lys192 of PolXBs, a residue moderately conserved in the palm subdomain of bacterial PolXs and immediately preceding two catalytic aspartates of the polymerization reaction. The results point to a function of residue Lys192 in guaranteeing the right orientation of the DNA substrates at the polymerization and histidinol phosphatase active sites. The results presented here and the recently solved structures of other bacterial PolX ternary complexes lead us to propose a structural model to account for the appropriate coordination of the different catalytic activities of bacterial PolXs.     

Crystal structure of enteropeptidase light chain complexed with an analog of the trypsinogen activation peptide.

Enteropeptidase is a membrane-bound serine protease that initiates the activation of pancreatic hydrolases by cleaving and activating trypsinogen. The enzyme is remarkably specific and cleaves after lysine residues of peptidyl substrates that resemble trypsinogen activation peptides such as Val-(Asp)4-Lys. To characterize the determinants of substrate specificity, we solved the crystal structure of the bovine enteropeptidase catalytic domain to 2.3 A resolution in complex with the inhibitor Val-(Asp)4-Lys-chloromethane. The catalytic mechanism and contacts with lysine at substrate position P1 are conserved with other trypsin-like serine proteases. However, the aspartyl residues at positions P2-P4 of the inhibitor interact with the enzyme surface mainly through salt bridges with the Nzeta atom of Lys99. Mutation of Lys99 to Ala, or acetylation with acetic anhydride, specifically prevented the cleavage of trypsinogen or Gly-(Asp)4-Lys-beta-naphthylamide and reduced the rate of inhibition by Val-(Asp)4-Lys-chloromethane 22 to 90-fold. For these reactions, Lys99 was calculated to account for 1.8 to 2.5 kcal mol(-1) of the free energy of transition state binding. Thus, a unique basic exosite on the enteropeptidase surface has evolved to facilitate the cleavage of its physiological substrate, trypsinogen.     

Formation of Nepsilon-(hexanonyl)lysine in protein exposed to lipid hydroperoxide. A plausible marker for lipid hydroperoxide-derived protein modification.

The objectives of this study were to estimate the structure of the lipid hydroperoxide-modified lysine residue and to prove the presence of the adducts in vivo. The reaction of lipid hydroperoxide toward the lysine moiety was investigated employing N-benzoyl-glycyl-L-lysine (Bz-Gly-Lys) as a model compound of Lys residues in protein and 13-hydroperoxyoctadecadienoic acid (13-HPODE) as a model of the lipid hydroperoxides. One of the products, compound X, was isolated from the reaction mixture of 13-HPODE and Bz-Gly-Lys and was then identified as N-benzoyl-glycyl-Nepsilon-(hexanonyl)lysine. To prove the formation of Nepsilon-(hexanonyl)lysine, named HEL, in protein exposed to the lipid hydroperoxide, the antibody to the synthetic hexanonyl protein was prepared and then characterized in detail. Using the anti-HEL antibody, the presence of HEL in the lipid hydroperoxide-modified proteins and oxidized LDL was confirmed. Furthermore, the positive staining by anti-HEL antibody was observed in human atherosclerotic lesions using an immunohistochemical technique. The amide-type adduct may be a useful marker for the lipid hydroperoxide-derived modification of biomolecules.     

Structural, kinetic and computational investigation of Vitis vinifera DHDPS reveals new insight into the mechanism of lysine-mediated allosteric inhibition

Lysine is one of the most limiting amino acids in plants and its biosynthesis is carefully regulated through inhibition of the first committed step in the pathway catalyzed by dihydrodipicolinate synthase (DHDPS). This is mediated via a feedback mechanism involving the binding of lysine to the allosteric cleft of DHDPS. However, the precise allosteric mechanism is yet to be defined. We present a thorough enzyme kinetic and thermodynamic analysis of lysine inhibition of DHDPS from the common grapevine, Vitis vinifera (Vv). Our studies demonstrate that lysine binding is both tight (relative to bacterial DHDPS orthologs) and cooperative. The crystal structure of the enzyme bound to lysine (2.4 Å) identifies the allosteric binding site and clearly shows a conformational change of several residues within the allosteric and active sites. Molecular dynamics simulations comparing the lysine-bound (PDB ID 4HNN) and lysine free (PDB ID 3TUU) structures show that Tyr132, a key catalytic site residue, undergoes significant rotational motion upon lysine binding. This suggests proton relay through the catalytic triad is attenuated in the presence of lysine. Our study reveals for the first time the structural mechanism for allosteric inhibition of DHDPS from the common grapevine.     

An arginine-aspartate network in the active site of bacterial TruB is critical for catalyzing pseudouridine formation

Pseudouridine synthases introduce the most common RNA modification and likely use the same catalytic mechanism. Besides a catalytic aspartate residue, the contributions of other residues for catalysis of pseudouridine formation are poorly understood. Here, we have tested the role of a conserved basic residue in the active site for catalysis using the bacterial pseudouridine synthase TruB targeting U55 in tRNAs. Substitution of arginine 181 with lysine results in a 2500-fold reduction of TruB’s catalytic rate without affecting tRNA binding. Furthermore, we analyzed the function of a second-shell aspartate residue (D90) that is conserved in all TruB enzymes and interacts with C56 of tRNA. Site-directed mutagenesis, biochemical and kinetic studies reveal that this residue is not critical for substrate binding but influences catalysis significantly as replacement of D90 with glutamate or asparagine reduces the catalytic rate 30- and 50-fold, respectively. In agreement with molecular dynamics simulations of TruB wild type and TruB D90N, we propose an electrostatic network composed of the catalytic aspartate (D48), R181 and D90 that is important for catalysis by fine-tuning the D48-R181 interaction. Conserved, negatively charged residues similar to D90 are found in a number of pseudouridine synthases, suggesting that this might be a general mechanism.     

Kinetics of chemical modification of arginine and lysine residues in calf thymus histone H1

The arginine and lysine residues of calf thymus histone H1 were modified with large molar excesses of 2,3-butanedione and O-methylisourea, respectively. Kinetic study of the modification reaction of the arginine residue revealed that the reaction is divided into the two pseudo-first-order processes. About a third (1 Arg) of the total arginine residues of the H1 molecule was rapidly modified without causing any detectable structural change of the molecule, and the slow modification of the remaining arginine residues (2 Arg) led to a loss of the folded structure of H1. In the case of lysine residue modification, 93% (56 Lys) of the total lysine residues of the H1 was modified with the same rate constant, while 7% (4 Lys) of lysine residue remained unmodified. When the reaction was performed in the presence of 6M guanidine-HCl, all of lysine residues were modified. It is concluded that the 2 arginine and 4 lysine residues resistant to modification are buried in interior regions of the H1 molecule and play an important role in the formation of the H1 globular structure, while the other 1 arginine and 56 lysine residues are exposed to solvent.     

A novel site for ubiquitination: the N-terminal residue, and not internal lysines of MyoD, is essential for conjugation and degradation of the protein.

The ubiquitin proteolytic pathway is a major system for selective protein degradation in eukaryotic cells. One of the first steps in the degradation of a protein via this pathway involves selective modification of epsilon-NH2 groups of internal lysine residues by ubiquitination. To date, this amino group has been the only known target for ubiquitination. Here we report that the N-terminal residue of MyoD is sufficient and necessary for promotion of conjugation and subsequent degradation of the protein. Substitution of all lysine residues in the protein did not affect significantly its conjugation and degradation either in vivo or in vitro. In cells, degradation of the lysine-less protein is inhibited by the proteasome inhibitors MG132 and lactacystin. Inhibition is accompanied by accumulation of high molecular mass ubiquitinated forms of the modified MyoD. In striking contrast, wild-type MyoD, in which all the internal Lys residues have been retained but the N-terminus has been extended by fusion of a short peptide, is stable both in vivo and in vitro. In a cell-free system, ATP and multiple ubiquitination are essential for degradation of the lysine-less protein. Specific chemical modifications have yielded similar results. Selective blocking of the alpha-NH2 group of wild-type protein renders it stable, while modification of the internal Lys residues with preservation of the free N-terminal group left the protein susceptible to degradation. Our data suggest that conjugation of MyoD occurs via a novel modification involving attachment of ubiquitin to the N-terminal residue. The polyubiquitin chain is then synthesized on an internal Lys residue of the linearly attached first ubiquitin moiety.     

Structural and functional roles of highly conserved serines in human lipoprotein lipase. Evidence that serine 132 is essential for enzyme catalysis

The structure of human lipoprotein lipase was recently deduced from its cDNA sequence. It contains 8 serine residues (residues 45, 132, 143, 172, 193, 244, 251, and 363) that are absolutely conserved in both lipoprotein lipase and hepatic lipase across all species studied. The high homology between lipoprotein lipase, hepatic lipase, and pancreatic lipase suggests that the catalytic functions of these enzymes share a common mechanism and that one of the 8 conserved serines in human lipoprotein lipase must play a catalytic role as does serine 152 in the case of pancreatic lipase (Winkler, F. K., D'Arcy, A., and Hunziker, W. Nature 343, 771-774). We expressed wild-type and site-specific mutants of human lipoprotein lipase in COS cells in vitro. We produced two to four substitution mutants involving each of the 8 serines and assayed a total of 22 mutants for both enzyme activity and the amount of immunoreactive enzyme mass produced. Immunoreactive lipase was detected in all cases. With the exception of Ser132, for each of the 8 serine mutants we studied, at least one of several mutants at each position showed detectable enzyme activity. All three substitution mutants at Ser132, Ser----Thr, Ser----Ala, and Ser----Asp, were totally inactive. Ser132 occurs in the consensus sequence Gly-Xaa-Ser-Xaa-Gly present in all serine proteinases and in human pancreatic lipase. The x-ray crystallography structure of human pancreatic lipase suggests that the analogous serine residue in human pancreatic lipase, Ser152, is the nucleophilic residue essential for catalysis. Our biochemical data strongly support the conclusion that Ser132 in human lipoprotein lipase is the crucial residue required for enzyme catalysis. The observed specific activities of the variants involving the other seven highly conserved serines in human lipoprotein lipase are consistent with the interpretation that this enzyme has a three-dimensional structure very similar to that of human pancreatic lipase.