Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Recent genetic mapping of the aspartokinase II (lysC) operon of Bacillus subtilis [M. Petricek. L. Rutberg & L. Hederstedt (1989) FEMS Microbiology Letters 61, 85-88; N.Y. Chen. J. J. Zhang & H. Paulus (1989) Journal of General Microbiology 135, 2931-2940] has shown its chromosomal location to be close to the aecA locus, the mutation of which leads to highly increased levels of aspartokinase II. In order to examine the relationship between lysC and aecA, we have cloned the control regions of the lysC operon from several independent aecA mutants and determined their nucleotide sequences. The nucleotide sequences of the aecA mutants differed from the wild-type sequence by the substitution of one or two nucleotides at two widely separated sites in the transcribed leader region of the lysC operon. To confirm that the observed nucleotide changes are indeed responsible for the AecA phenotype and not simply the reflection of sequence polymorphisms in different B. subtilis strains, we introduced the same nucleotide substitutions as those observed in the aecA strains into the leader region of the wild-type lysC operon by oligonucleotide-directed mutagenesis. The expression of the mutagenized genes was analysed after transcriptional or translational fusion to lacZ in a single-copy integration vector. The levels of beta-galactosidase were greatly elevated by the nucleotide substitutions, with similar increases observed in transcriptional and translational fusions. The high level of expression of beta-galactosidase in the lysC'-lac'Z strains with nucleotide substitutions corresponding to the aecA mutations was resistant to repression by L-lysine but was completely abolished by the inactivation of the lysC promoter.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Identification of aecA mutations in Bacillus subtilis as nucleotide substitutions in the untranslated leader region of the aspartokinase II operon. 190 38

Site-directed substitutions (Asp, Gly, Gln, His, and Lys) were made for Glu-461 of beta-galactosidase (Escherichia coli). All substitutions resulted in loss of most activity. Substrates and a substrate analog inhibitor were bound better by the Asp-substituted enzyme than by the normal enzyme, about the same for enzyme substituted with Gly, but only poorly when Gln, His, or Lys was substituted. This shows that Glu-461 is involved in substrate binding. Binding of the positively charged transition state analog 2-aminogalactose was very much reduced with Gly, Gln, His, and Lys, whereas the Asp-substituted enzyme bound this inhibitor even better than did the wild-type enzyme. Since Asp, like Glu, is negatively charged, this strongly supports the proposal that one role of Glu-461 is to electrostatically interact with a positively charged galactosyl transition state intermediate. The substitutions also affected the ability of the enzyme to bind L-ribose, a planar analog of D-galactose that strongly inhibits beta-galactosidase activity. This indicates that the binding of a planar "galactose-like" compound is somehow mediated through Glu-461. The data indicated that the presence of Glu-461 is highly important for the acid catalytic component of kappa 2 (glycosylic bond cleavage or "galactosylation"), and therefore Glu-461 must be involved in a concerted acid catalytic reaction, presumably by stabilizing a developing carbonium ion. The kappa 2 values with o- and p-nitrophenyl-beta-D-galactopyranoside as substrates varied more or less as did the K8 values, indicating that most of the glycolytic bond breaking activity found for the enzymes from the mutants with these substrates was probably a result of strain or other such effects. The kappa 3 values (hydrolysis or "degalactosylation") of the substituted enzymes were also low, indicating that Glu-461 is important for that part of the catalysis. The enzyme with His substituted for Glu-461 had the highest kappa 3 value. This is probably a result of the formation of a covalent bond between His and the galactosyl part of the substrate.
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PMID:Determination of the roles of Glu-461 in beta-galactosidase (Escherichia coli) using site-specific mutagenesis. 196 5

A thermophilic and thermostable beta-galactosidase activity was purified to homogeneity from crude extracts of the archaebacterium Sulfolobus solfataricus, by a procedure including ion-exchange and affinity chromatography. The homogeneous enzyme had a specific activity of 116.4 units/mg at 75 degrees C with o-nitrophenyl beta-galactopyranoside as substrate. Molecular mass studies demonstrated that the S. solfataricus beta-galactosidase was a tetramer of 240 +/- 8 kDa composed of similar or identical subunits. Comparison of the amino acid composition of beta-galactosidase from S. solfataricus with that from Escherichia coli revealed a lower cysteine content and a lower Arg/Lys ratio in the thermophilic enzyme. A rabbit serum, raised against the homogeneous enzyme did not cross-react with beta-galactosidase from E. coli. The enzyme, characterized for its reaction requirements and kinetic properties, showed a thermostability and thermophilicity notably greater than those reported for beta-galactosidases from other mesophilic and thermophilic sources.
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PMID:Thermostable beta-galactosidase from the archaebacterium Sulfolobus solfataricus. Purification and properties. 210 16

The Mg2+ concentrations required for half maximal activity, the dissociation constants, and the free energies of binding for Mg2+ bound to wild type beta-galactosidase and several site specific mutants are reported. The mutants have one of the following substitutions: Glu-461 substituted with Asp, Gln, Gly, His, or Lys; or Tyr-503 substituted with Phe, His or Cys. Substitutions for Tyr-503 had little effect on the affinity of the enzyme for Mg2+, implying that Tyr-503 is not involved in Mg2+ binding. Neutrally charged amino acids substituted for the negatively charged Glu-461 significantly decreased the affinity of the enzyme for Mg2+ and substitution of positively charged amino acids at this position further decreased the affinity. On the other hand, substitution by Asp (negative charge) at position 461 had no effect on the binding. Thus, the negatively charged side chain of Glu-461 is important for divalent cation binding to beta-galactosidase.
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PMID:Site specific mutants of beta-galactosidase show that Tyr-503 is unimportant in Mg2+ binding but that Glu-461 is very important and may be a ligand to Mg2+. 211 47

Tyr-503 of beta-galactosidase was specifically replaced with Phe, His, Cys, and Lys using site-directed mutagenesis. The normal enzyme and the substituted enzymes were purified. The activities of each of the substituted enzymes with o-nitrophenyl-beta-D-galactopyranoside (ONPG) and p-nitrophenyl-beta-D-galactopyronoside (PNPG) were very low and Y503K-beta-galactosidase was essentially inactive, showing that Tyr-503 is important for activity. The stability (including tetrameric stability) of the enzymes at 4 and 25 degrees C was essentially the same as that of the wild-type enzyme and the cleavage patterns on sodium dodecyl sulfate gels after protease action were unchanged. These studies thus indicate that Tyr-503 has no noticeable influence on stability under normal conditions. The substitutions for Tyr-503 had some small effects on the binding of both substrate and inhibitor. However, both kappa 2 (glycosidic bond cleavage rate) and kappa 3 (hydrolysis rate constant) were dramatically reduced. Each substitution except that of Lys (which can be explained by electrostatic effects) gave decreases in kappa 2 and kappa 3 of roughly the same magnitude regardless of whether the substitutions were conservative or not. This strongly implies that the changes in rate were not due to conformational changes as it is very unlikely that there would be such similar decreases in the values of kappa 2 and kappa 3 for amino acids with such different structures and chemical properties if the changes in rate were due to conformational differences. The data suggest that one possible role of Tyr-503 is as a general acid/base catalyst. Profiles of the kinetic data of the enzymes as functions of pH supported the suggestion that Tyr-503 normally acts as a general acid and base catalyst. When Tyr-503 was substituted by His, a small amount of base catalytic activity seemed to be restored. The strongest evidence that Tyr-503 acts as an acid catalyst came from studies with isoquinolinium-beta-D-galactopyranoside as the substrate. The kappa cat(s) of Y503F-beta-galactosidase and of Y503C-beta-galactosidase decreased by about an order of magnitude while the rate decreases were about 3 orders of magnitude with ONPG and PNPG. The breakdown of isoquinolinium-beta-D-galactopyranoside cannot be catalyzed by acids.
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PMID:Multiple replacements establish the importance of tyrosine-503 in beta-galactosidase (Escherichia coli). 212 20

Marine mussels secrete the byssus in order to attach to solid surfaces and to survive under the turbulent effects of waves. The adhesive responsible for this attachment is the polyphenolic protein secreted by the phenol gland in the foot of the animal. To purify this adhesive protein from the chilean mussel Mylilus chilensis, a modification of previous procedures has been developed. Accordingly, the protein is differentially precipitated with acetone in the presence of 0.25 N HCl. The purified protein is rich in the amino acids lysine, 3,4-dihydroxyphenylalanine, serine, threonine, proline and hydroxyproline. The protein exhibited strong adhesion to glass and other solid supports. Moreover, it has been found that the adhesive protein can mediate the immobilization of beta-galactosidase to glass. About 75% of the enzyme activity was immobilized under the experimental conditions described. This is the first study reporting the use of the polyphenolic protein to immobilize enzymes.
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PMID:Bioadhesives: a biotechnological opportunity. 213 19

The proteolytic targeting function of ubiquitin was investigated by a combination of site-specific mutagenesis and covalent modification. Lys48 was replaced by a cysteine via mutagenesis of a synthetic ubiquitin gene to generate the mutant Ub-C48. The single cysteine residue in Ub-C48 can be converted into a lysine analog by modification with the sulfhydryl-specific reagent, aminoethyl-8 (N-(iodoethyl)trifluoroacetamide). The resulting protein, Ub-(S-aminoethyl)C48, is equivalent to a wild type ubiquitin except for the substitution of a sulfur atom at the gamma carbon of Lys48. We have tested the ability of these two modified ubiquitins to target the degradation of an engineered beta-galactosidase substrate protein in ubiquitin-depleted reticulocyte lysates. Ub-C48 was unable to stimulate the degradation of this protein substrate although a monoubiquitinated beta-galactosidase was formed. In contrast, Ub-(S-aminoethyl)C48 appears to be as effective as wild type ubiquitin in targeting this substrate protein's degradation as well as the formation of multiply ubiquitinated beta-galactosidase intermediates. In conjunction with the cysteine substitution and modification, we have also examined the effects of blocking the amino groups in ubiquitin with reductive methylation. The methylation of either Lys48 in ubiquitin or its S-aminoethylcysteine counterpart abolished its proteolytic function while the blockage of the remaining six lysines in Ub-(S-aminoethyl)C48 did not alter its competence. Thus, of the seven lysine residues in ubiquitin, only Lys48 is essential. These results established unambiguously that a uniform multiubiquitin chain with ubiquitin-ubiquitin linkage solely at Lys48 is sufficient to target the degradation of a substrate protein in ubiquitin-mediated proteolysis.
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PMID:A uniform isopeptide-linked multiubiquitin chain is sufficient to target substrate for degradation in ubiquitin-mediated proteolysis. 216 Apr 52

A run of 11 adenine or thymine residues at the 5' end of an out-of-frame lacZ gene causes a high level of beta-galactosidase expression in E. coli. This effect was not observed for a run of guanine residues. Reverse transcription of mRNA isolated from E. coli containing the run of 11 A's reveals heterogeneity of transcript length while reverse transcription of mRNA isolated from S. cerevisiae containing the same gene shows no heterogeneity. Protein sequencing of the beta-galactosidase molecules derived from the out-of-frame construct containing a run of adenines reveals the addition of a lysine at the run. A new method was developed where messages small enough to allow resolution of single nucleotide differences on an acrylamide gel are electrophoresed, electroblotted onto nylon and probed. This confirmed the reverse transcription results and showed that additional residues can be added to transcripts derived from DNA containing 10 or 11 thymine residues. A mechanism for slippage is discussed where the A-U rich RNA-DNA hybrid can denature during elongation and rehybridize in an offset position, causing the addition of extra residues to the transcript.
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PMID:Transcriptional slippage occurs during elongation at runs of adenine or thymine in Escherichia coli. 219 64

The binding site of a monoclonal antibody to the haemagglutinin-neuraminidase (HN) polypeptide of Newcastle disease virus (NDV) has been located. Complementary DNA or synthetic oligonucleotides corresponding to portions of the HN gene were cloned into the Escherichia coli vector pUC19 and fragments of the HN protein were thereby fused to the alpha-peptide of beta-galactosidase. Western blot analysis of E. coli lysates containing expressed fragments of the HN cDNA or synthetic oligonucleotides identified an antibody-binding peptide (Asp-Glu-Gln-Asp-Tyr-Gln-Ile-Arg; amino acid residues 346 to 353). Nucleotide sequence analysis of an antibody-resistant mutant of NDV revealed a Glu (wild-type) to Lys (mutant) substitution within the above sequence. The methods described could be useful for the location of continuous epitopes of other polypeptides.
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PMID:Location of a neutralizing epitope for the haemagglutinin-neuraminidase glycoprotein of Newcastle disease virus. 245 68

Our previous work has shown that, in the yeast Saccharomyces cerevisiae, any of the eight stabilizing amino-terminal residues confers a long (greater than 20 h) half-life on a test protein beta-galactosidase (beta gal), whereas 12 destabilizing amino-terminal residues confer on beta gal half-lives from less than 3 min to 30 min. We now show that an analogous single-residue code (the N-end rule) operates in an in vitro system derived from mammalian reticulocytes. We also show that the N-end rule has a hierarchical structure. Specifically, amino-terminal Glu and Asp (and also Cys in reticulocytes) are secondary destabilizing residues in that they are destabilizing through their ability to be conjugated to primary destabilizing residues such as Arg. Amino-terminal Gln and Asn are tertiary destabilizing residues in that they are destabilizing through their ability to be converted, via selective deamidation, into secondary destabilizing residues Glu and Asp. Furthermore, in reticulocytes, distinct types of the N-end-recognizing activity are shown to be specific for three classes of primary destabilizing residues: basic (Arg, Lys, His), bulky hydrophobic (Phe, Leu, Trp, Tyr), and small uncharged (Ala, Ser, Thr). Features of the N-end rule in reticulocytes suggest that the exact form of the N-end rule may depend on the cell's physiological state, thereby providing a mechanism for selective destruction of preexisting proteins upon cell differentiation.
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PMID:Universality and structure of the N-end rule. 250 81


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