Analysis of essential histidine residues of maize branching enzymes by chemical modification and site-directed mutagenesis

The association between antibody reactivity to the neutralizing epitope ELDKWA in the transmembrane glycoprotein gp41 and disease progression was investigated in 29 children perinatally infected with HIV-1. Levels of antibody reactivity to this epitope, measured over time, were associated with absolute CD4+ lymphocyte numbers and disease status, and inversely associated with the levels of acid-dissociated p24 antigen in the plasma. Early virus isolates from 10 of 12 children with no detectable antibody reactivity to this epitope were sequenced. Only three contained sequences that differed from the consensus, indicating that this epitope is well conserved in this population. None of these three children developed antibodies to the autologous sequences, indicating that at least 80% of children with negative antibody reactivity to this epitope were true nonresponders. Together, these results indicate that the ELDKWA determinant could be an important component in the formulation of a vaccine or for immunotherapeutic approaches to HIV-1 infection.     

Glycine residues provide flexibility for enzyme active sites

The high resolution refined structures of 23 enzymes were analyzed to determine the properties of amino acids involved in active site regions. These regions were found to be rich in G-X-Y or Y-X-G oligopeptides, where X and Y are polar and non-polar residues, respectively, that are small and with low polarity. Other regions of the enzyme molecules have significantly fewer of these sequences. These features suggest that glycine residues may provide flexibility necessary for enzyme active sites to change conformation, and the G-X-Y or Y-X-G oligopeptides may be a motif for the formation of enzyme active sites.     

Identification of cysteine and arginine residues essential for the phosphotransacetylase from Methanosarcina thermophila

Phosphotransacetylase catalyzes the following reaction: CoASH + CH3CO2PO3(2-) <==> CH3COSCoA + HPO4(2-) (where CoA is coenzyme A). Based on biochemical characterization of the enzyme from the obligate anaerobe Clostridium kluyveri, a ternary mechanism was proposed in which an unspecified cysteine abstracts a proton from CoASH forming a nucleophilic thiolate anion which attacks acetyl phosphate (J. Henkin and R. H. Abeles, Biochemistry 15:3472-3479, 1976). Heterologous production in Escherichia coli of the phosphotransacetylase from Methanosarcina thermophila, an obligately anaerobic methanoarchaeon, allowed site-specific replacements to identify essential residues. All four cysteines present in the sequence were individually replaced with alanine, and the kinetic constants of the altered enzymes were determined. The results indicated that only C159 is essential for activity; however, replacement with serine resulted in a fully active enzyme. Activity of the unaltered phosphotransacetylase was sensitive to N-ethylmaleimide. Inhibition kinetics of altered enzymes indicated that this sensitivity resulted from modification of C312, which is at the active site but itself is nonessential for catalysis. Five arginines were individually replaced with glutamine. Kinetic analysis of the altered enzymes identified R310 as essential for activity. Of the four nonessential for activity, R87 and R133 appear to be involved in binding CoA.     

Functional residues at the active site of bovine brain adenosine deaminase

Brain adenosine deaminase was investigated in order to identify amino acid residues essential for its catalytic activity. The pH dependence of log Vmax shows that the enzyme activity depends on two ionizing groups with pK values of 5.4, that must be unprotonated, and 8.4, that must be protonated, for the catalysis. These same groups are observed in the Vmax/Km profiles. The plausible role of histidine residues at the active site of brain adenosine deaminase was proved by chemical modification with (DEP). The histidine specific reagent inactivated the enzyme following a pseudo first-order kinetics with a second-order rate constant of 8.9 10(-3) (+/- 1.8 10(-3)) M-1 min-1. The inhibition of the enzyme with PCMBS was studied monitoring the enzyme activity after incubation with the inhibitor. Brain adenosine deaminase exhibited a characteristic intrinsic tryptophan fluorescence with an emission peak centered at 335 nm. Stern-Volmer quenching parameters in the presence of acrylamide and iodide indicated that tryptophan residues are buried in the native molecule. Tryptophan residues also showed a high heterogeneity that was increased after binding of ground- and transition-state analogs to the enzyme.     

Determining the number of essential ligand binding sites in enzymes using the slopes of the Wang-Srivastava plots

Systemic necrotizing vasculitis is uncommon in children and may be rarely associated with gangrene. We describe a 3-yr-old girl with parvovirus B19-induced necrotizing vasculitis whose digital gangrene was successfully treated with iloprost, a prostacyclin analogue.     

The active site of hamster 3-hydroxy-3-methylglutaryl-CoA reductase resides at the subunit interface and incorporates catalytically essential acidic residues from separate polypeptides

We employed site-directed mutagenesis based on sequence comparisons and characterization of purified mutant enzymes to identify Glu558 and Asp766 of Syrian hamster 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34) as essential for catalysis. Mutant enzymes E558D, E558Q, and D766N had wild-type Km values for (S)-HMG-CoA and NADPH, but exhibited less than 0.5% of the wild-type catalytic activity. The inactive mutant polypeptides E558Q and D766N nevertheless can associate to generate an active enzyme. In vitro, 6% of the wild-type activity was observed when mutant polypeptides E558D and D766N were mixed in the absence of chaotropic agents. When mutant polypeptides E558Q and D766N were co-expressed in Escherichia coli, the resulting purified enzyme had 25% of wild-type activity. Hamster HMG-CoA reductase thus is a two-site, dimeric enzyme whose subunits associate to form an active site in which each monomer contributes at least one residue (e.g. Glu558 from one monomer and Asp766 from the other). The wild-type enzyme behaves as a dimer during size exclusion chromatography and has one HMG-CoA binding site per monomer. Syrian hamster HMG-CoA reductase thus appears to be a homodimer with two active sites which are located at the subunit interface.     

Site-directed mutagenesis of putative active site residues of MunI restriction endonuclease: replacement of catalytically essential carboxylate residues triggers DNA binding specificity

Mapping of the conserved sequence regions in the restriction endonucleases MunI (C/AATTG) and EcoRI (G/AATTC) to the known X-ray structure of EcoRI allowed us to identify the sequence motif 82PDX14EXK as the putative catalytic/Mg2+ ion binding site of MunI [Siksnys, V., Zareckaja, N., Vaisvila, R., Timinskas, A., Stakenas, P., Butkus, V., & Janulaitis, A. Gene (1994) 142, 1-8]. Site-directed mutagenesis was then used to test whether amino acids P82, D83, E98, and K100 were important for the catalytic activity of MunI. Mutation P82A generated only a marginal effect on the cleavage properties of the enzyme. Investigation of the cleavage properties of the D83, E98, and K100 substitution mutants, however, in vivo and in vitro, revealed either an absence of catalytic activity or markedly reduced catalytic activity. Interestingly, the deleterious effect of the E98Q replacement in vitro was partially overcome by replacement of the metal cofactor used. Though the catalytic activity of the E98Q mutant was only 0.4% of WT under standard conditions (in the presence of Mg2+ ions), the mutant exhibited 40% of WT catalytic activity in buffer supplemented with Mn2+ ions. Further, the DNA binding properties of these substitution mutants were analyzed using the gel shift assay technique. In the absence of Mg2+ ions, WT MunI bound both cognate DNA and noncognate sequences with similar low affinities. The D83A and E98A mutants, in contrast, in the absence of Mg2+ ions, exhibited significant specificity of binding to cognate DNA, suggesting that the substitutions made can simulate the effect of the Mg2+ ion in conferring specificity to the MunI restriction enzyme.     

The five cysteine residues located in the active site region of bovine aspartyl (asparaginyl) beta-hydroxylase are not essential for catalysis

In previous chemical modification studies on bovine aspartyl (asparaginyl) beta-hydroxylase, cysteines were implicated as critical catalytic residues. Using site-directed mutagenesis, the five cysteine residues located in a highly conserved region of the enzyme identified as the active site were individually mutated to alanine. Substitutions at cysteine 637, 644, 656, 681, and 696 resulted in active mutant enzymes indicating that these residues are not required for catalysis.     

Covalent catalysis in nucleotidyl transfer. A KTDG motif essential for enzyme-GMP complex formation by mRNA capping enzyme is conserved at the active sites of RNA and DNA ligases

Vaccinia virus RNA capping enzyme, a heterodimer of 95- and 31-kDa subunits, catalyzes transfer of GMP from GTP to the 5'-diphosphate terminus of RNA via a covalent enzyme-guanylate intermediate. The GMP residue is attached to the 95-kDa subunit through a phosphoamide bond to the epsilon-amino group of a lysine residue. The amino acid sequence of the large subunit includes a lysine-containing motif, Tyr-X-X-X-Lys260-Thr-Asp-Gly, that is conserved in the RNA guanylyltransferases encoded by Shope fibroma virus and Saccharomyces cerevisiae. The KXDG motif is also encountered at the sites of covalent adenylylation of bacteriophage T4 RNA ligase and mammalian DNA ligase I (Thogerson, H. C., Morris, H. R., Rand, K. N., and Gait, M. J. (1985) Eur. J. Biochem. 147, 325-329; Tomkinson, A. E., Totty, N. F., Ginsburg, M., and Lindahl, T. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 400-404). We find that conservative amino acid substitutions at three out of four positions within the KTDG sequence of vaccinia capping enzyme either prevent or strongly inhibit enzyme-guanylate formation. The conserved motif is therefore an essential component of the guanylyltransferase domain. Lys260 is implicated as the active site. Comparison of the sequences of capping enzymes and polynucleotide ligases from diverse sources suggests that KX(D/N)G may be a signature element for covalent catalysis in nucleotidyl transfer.     

Isolation and characterization of a dibasic selective metalloendopeptidase from rat testes that cleaves at the amino terminus of arginine residues

A metalloendopeptidase that selectively cleaves doublets of basic amino acids on the amino-terminal side of arginine residues was purified to homogeneity from rat testes and analyzed further. Two catalytically active forms with apparent relative molecular masses of 110,000 and 140,000 Da, respectively, were present in the purified preparation of the enzyme. Antibodies raised against the purified testis endopeptidase revealed by immunoblot both the 110- and 140-kDa forms in both rat testis and brain cortex extracts. The isolated enzyme was inhibited by metal chelators and divalent cations. Its activity, lost after preincubation with EDTA, was restored by low concentrations of Zn2+ and Mn2+, thus demonstrating the metallopeptidase nature of the enzyme. This endopeptidase also exhibited a high sensitivity to amastatin (100% inhibition at 20 microM), an aminopeptidase inhibitor. A substrate specificity study using physiologically important or synthetic peptides containing a processing dibasic site indicated that cleavage occurred selectively at the amino-terminal side of an arginine residue, independent of the nature of the basic doublet. The enzyme produced such a cleavage at the Arg-Lys doublet of somatostatin 28 (Km = 43 microM), at the Arg-Arg doublet of dynorphin A (Km = 6.45 microM) and atrial natriuretic factor (Km = 6.25 microM), and at the Lys-Arg doublet of preproneurotensin-(154-170) (Km = 17.3 microM). Moreover, cleavage efficiency was found to be higher for the larger substrates. The distinctive properties of this endopeptidase imply that this protein is a member of a novel class of proteolytic enzymes that may be involved in the endoproteolytic maturation of hormonal precursors.     

The epitopes targeted by the rheumatoid arthritis-associated antifilaggrin autoantibodies are posttranslationally generated on various sites of (pro)filaggrin by deimination of arginine residues

Antifilaggrin autoantibodies (AFA) are a population of IgG autoantibodies associated to rheumatoid arthritis (RA), which includes the so-called "antikeratin" Abs and antiperinuclear factor. AFA are the most specific serological markers of RA. We previously showed that they recognize human epidermal filaggrin and other profilaggrin-related proteins of various epithelial tissues. Here, we report further characterization of the protein Ags and epitopes targeted by AFA. All the Ags that exhibit numerous neutral/ acidic isoelectric variants were immunochemically demonstrated to be deiminated proteins. In vitro deimination of a recombinant human filaggrin by a peptidylarginine deiminase generated AFA epitopes on the protein. Moreover, two of three filaggrin-derived synthetic peptides with a citrulline in the central position were specifically and widely recognized by AFA affinity-purified from a series of RA sera. These results indicate that citrulline residues are constitutive of the AFA epitopes, but only in the context of specific amino acid sequences of filaggrin. In competition experiments, the two peptides abolished the AFA reactivity of RA sera, showing that they present major AFA epitopes. These data should help in the identification of a putative deiminated AFA-inducing or cross-reactive articular autoantigen and provide new insights into the pathogenesis of RA. They could also open the way toward specific immunosuppressive and/or preventive therapy of RA.     

The conserved asparagine and arginine are essential for catalysis of mammalian adenylyl cyclase

Mammalian adenylyl cyclases have two homologous cytoplasmic domains (C1 and C2), and both domains are required for the high enzymatic activity. Mutational and genetic analyses of type I and soluble adenylyl cyclases suggest that the C2 domain is catalytically active and the C1 domain is not; the role of the C1 domain is to promote the catalytic activity of the C2 domain. Two amino acid residues, Asn-1025 and Arg-1029 of type II adenylyl cyclase, are conserved among the C2 domains, but not among the C1 domains, of adenylyl cyclases with 12 putative transmembrane helices. Mutations at each amino acid residue alone result in a 30-100-fold reduction in Kcat of adenylyl cyclase. However, the same mutations do not affect the Km for ATP, the half-maximal concentration (EC50) for the C2 domain of type II adenylyl cyclase to associate with the C1 domain of type I adenylyl cyclase and achieve maximal enzyme activity, or the EC50 for forskolin to maximally activate enzyme activity with or without Gsalpha. This indicates that the mutations at these two residues do not cause gross structural alteration. Thus, these two conserved amino acid residues appear to be crucial for catalysis, and their absence from the C1 domains may account for its lack of catalytic activity. Mutations at both amino acid residues together result in a 3,000-fold reduction in Kcat of adenylyl cyclase, suggesting that these two residues have additive effects in catalysis. A second site suppressor of the Asn-1025 to Ser mutant protein has been isolated. This suppressor has 17-fold higher activity than the mutant and has a Pro-1015 to Ser mutation.     

Evidence for two nonidentical drug-interaction sites in the human P-glycoprotein

Human P-glycoprotein (Pgp) confers multidrug resistance to cancer cells by ATP-dependent extrusion of a great many structurally dissimilar hydrophobic compounds. The manner in which Pgp recognizes these different substrates is unknown. The protein shows internal homology between its N- and C-terminal halves, each comprised of six putative transmembrane helices and a consensus ATP binding/utilization site. Photoactive derivatives of certain Pgp substrates specifically label two regions, one on each half of the protein. In this study, using [125I]iodoarylazidoprazosin ([125I]IAAP), a photoactive analog of prazosin, we have demonstrated the presence of two nonidentical drug-interaction sites within Pgp. Taking advantage of a highly susceptible trypsin cleavage site in the linker region of Pgp, we characterized the [125I]IAAP binding to the N- and C-terminal halves. cis(Z)-Flupentixol, a modulator of Pgp function, preferentially increased the affinity of [125I]IAAP for the C-terminal half of the protein (C-site) by reducing the Kd from 20 to 6 nM without changing the labeling or affinity (Kd = 42-46 nM) of the N-terminal half (N-site). Also, the concentration of vinblastine (Pgp substrate) and cyclosporin A (Pgp modulator) required for 50% inhibition of [125I]IAAP binding to the C-site was increased 5- to 6-fold by cis(Z)-flupentixol without any effect on the N-site. In addition, [125I]IAAP binding to the N-site was less susceptible than to C-site to inhibition by vanadate which blocks ATP hydrolysis and drug transport. These data demonstrate the presence of at least two nonidentical substrate interaction sites in Pgp.     

Physical association of starch biosynthetic enzymes with starch granules of maize endosperm. Granule-associated forms of starch synthase I and starch branching enzyme II

Antibodies were used to probe the degree of association of starch biosynthetic enzymes with starch granules isolated from maize (Zea mays) endosperm. Graded washings of the starch granule, followed by release of polypeptides by gelatinization in 2% sodium dodecyl sulfate, enables distinction between strongly and loosely adherent proteins. Mild aqueous washing of granules resulted in near-complete solubilization of ADP-glucose pyrophosphorylase, indicating that little, if any, ADP-glucose pyrophosphorylase is granule associated. In contrast, all of the waxy protein plus significant levels of starch synthase I and starch branching enzyme II (BEII) remained granule associated. Stringent washings using protease and detergent demonstrated that the waxy protein, more than 85% total endosperm starch synthase I protein, and more than 45% of BEII protein were strongly associated with starch granules. Rates of polypeptide accumulation within starch granules remained constant during endosperm development. Soluble and granule-derived forms of BEII yielded identical peptide maps and overlapping tryptic fragments closely aligned with deduced amino acid sequences from BEII cDNA clones. These observations provide direct evidence that BEII exits as both soluble and granule-associated entities. We conclude that each of the known starch biosynthetic enzymes in maize endosperm exhibits a differential propensity to associate with, or to become irreversibly entrapped within, the starch granule.     

Essential histidyl residues at the active site(s) of sucrose-phosphate synthase from Prosopis juliflora

Antimicrobial resistance in nosocomial isolates is of increasing concern to the clinician, particularly in intensive care units. With more expensive drugs and prolonged periods of hospitalization required, resistance can result in increased healthcare costs. For the patient, infection with multiply resistant strains of bacteria is associated with high mortality rates. This review focuses on the prevalence of nosocomial infections throughout Europe, with particular emphasis on the prevalence of resistance to common antimicrobial agents. The beta-lactams are the most frequently prescribed antimicrobials, and the growing importance of extended spectrum beta-lactamases and the hyperproduction of chromosomal beta-lactamase by stably derepressed mutants in the development of microbial resistance are discussed. Given that the most common reason for modification of an initial empiric antibiotic treatment is the isolation of microorganisms not susceptible to the initial choice of treatment, the results from two European multicenter trials comparing the efficacy of the carbapenems, meropenem, and imipenem/cilastatin, for the treatment of serious nosocomial infections, are appraised. In light of these results, it can be concluded that the carbapenems are effective as initial empiric monotherapy for nosocomial infections because of their broad spectrum of efficacy and stability to beta-lactamases.