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:3.2.1.26 (invertase)
4,927 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A carboxylate group occurs in each of the active sites of the intestinal sucrase-isomaltase complex and can be labeled selectively by [3H]conduritol-B-epoxide (Quaroni, A., Gershon, E., and Semenza, G. (1974) J. Biol. Chem. 249, 6424-6433). After peptic digestion of the labeled, denatured, reduced, and cyanoethylated enzyme three radioactive peptides were isolated. The peptide originating from the sucrase subunit had the following sequence: Ile-Asp-Met-Asn-Gln-Pro-Asn-Ser-Ser; the other two, deriving from the isomaltase subunit, had the sequences: Asn-Gly2-Gln-Ile-Asp-Met and Ile-Asp-Met. The radioactive label was in each case bound to the beta carboxyl group of an aspartic acid residue.
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PMID:Partial amino acid sequences around the essential carboxylate in the active sites of the intestinal sucrase-isomaltase complex. 77 63

In the present communication molecular genetic approaches have been utilized to confirm the nature of the catalytic site of Streptococcus mutans glucosyltransferases (GTF)s. Site-directed mutagenesis was used to convert the putative sucrose binding Asp-451 of the GTF-I enzyme from S. mutans GS5 to Glu, Asn, and Thr. All three of the resulting mutated enzymes displayed no detectable sucrase or GTF activities. By contrast, mutation of nearby Asp residues did not markedly reduce enzymatic activity. The inactive enzymes also appear to bind acceptor dextrans as well as the parental enzyme. These results confirm the essential role of Asp-451 of the GTF-I from strain GS5 and analogous Asp residues in other related GTFs in enzymatic activity.
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PMID:Molecular genetic analysis of the catalytic site of Streptococcus mutans glucosyltransferases. 147 27

We have investigated different leader sequences for their ability to direct the efficient secretion of human epidermal growth factor (hEGF) from Saccharomyces cerevisiae. We designed a consensus signal sequence which directs secretion of hEGF from yeast as efficiently as the yeast invertase signal sequence. However, secretion is increased over fivefold by the introduction, after the signal sequence, of a synthetic 19-amino acid (aa) pro-sequence containing a cleavage recognition site for the KEX2 protease. Even in the absence of an Asn-linked glycosylation site, secretion of hEGF using the synthetic prepro-leader was as efficient as that directed by the alpha-factor leader. The role of the KEX2 protease cleavage site was investigated by mutation of the yeast alpha-factor KEX2 site (cleavage after Lys-Arg). Cleavage was obtained with the following order of efficiency, Lys-Arg greater than Pro-Arg greater than Asp-Arg, although the sequence context was also found to affect efficiency.
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PMID:Secretion of human epidermal growth factor from Saccharomyces cerevisiae using synthetic leader sequences. 193 55

Deglycosylated yeast invertase is irreversibly inactivated by conduritol B epoxide (CBE), an active-site-directed reagent. The inactivated enzyme contained 0.8 mol of CBE/mol of invertase monomer suggesting that the inactivation results from the modification of a single amino acid residue. Peptic digestion of [3H]CBE-labeled invertase followed by reverse phase column chromatography yielded two labeled peptides, both located at the amino-terminal end of the enzyme. Sequence analyses of these peptides revealed that Asp-23 is the modified residue. The role of Asp-23 in the catalytic process was investigated by changing it to Asn using site-directed mutagenesis of the SCU2 gene. The mutant enzyme was basically inactive, confirming a role for Asp-23 in the catalytic process.
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PMID:Identification of an active-site residue in yeast invertase by affinity labeling and site-directed mutagenesis. 211 24

Lysosomal alpha-glucosidase (acid maltase) is essential for degradation of glycogen in lysosomes. Enzyme deficiency results in glycogenosis type II. The amino acid sequence of the entire enzyme was derived from the nucleotide sequence of cloned cDNA. The cDNA comprises 3636 nt, and hybridizes with a messenger RNA of approximately 3.6 kb, which is absent in fibroblasts of two patients with glycogenosis type II. The encoded protein has a molecular mass of 104.645 kd and starts with a signal peptide. Sites of proteolytic processing are established by identification of N-terminal amino acid sequences of the 110-kd precursor, and the 76-kd and 70-kd mature forms of the enzyme encoded by the cDNA. Interestingly, both amino-terminal and carboxy-terminal processing occurs. Sites of sugar-chain attachment are proposed. A remarkable homology is observed between this soluble lysosomal alpha-glucosidase and the membrane-bound intestinal brush border sucrase-isomaltase enzyme complex. It is proposed that these enzymes are derived from the same ancestral gene. Around the putative active site of sucrase and isomaltase, 10 out of 13 amino acids are identical to the corresponding amino acids of lysosomal alpha-glucosidase. This strongly suggests that the aspartic acid residue at this position is essential for catalytic function of lysosomal alpha-glucosidase.
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PMID:Primary structure and processing of lysosomal alpha-glucosidase; homology with the intestinal sucrase-isomaltase complex. 304 72

1. Growth of a biotin-requiring strain of Saccharomyces cerevisiae in a medium containing a suboptimum concentration of biotin for growth caused a decreased synthesis of ornithine carbamoyltransferase as compared with yeast grown in a medium containing an optimum concentration of biotin. Inclusion of the biotin homologues norbiotin or homobiotin, but not bishomobiotin, in the biotin-deficient medium caused an appreciable increase in ornithine carbamoyltransferase synthesis without affecting growth or synthesis of total RNA and protein. The addition of norbiotin to biotin-deficient medium had no effect on the respiratory activity of the yeast or on the synthesis of aspartate carbamoyltransferase, acid phosphatase, beta-fructofuranosidase or malate dehydrogenase. 2. Synthesis of acetylornithine deacetylase and acetylornithine acetyltransferase was slightly diminished by the imposition of biotin deficiency, but the effect was not as great as on ornithine carbamoyltransferase synthesis. Incorporation of norbiotin in the biotin-deficient medium had no marked effect on the synthesis of any other arginine-pathway enzyme except ornithine carbamoyltransferase. 3. l-Ornithine induced synthesis of ornithine carbamoyltransferase in yeast grown in biotin-deficient medium, but in yeast grown in this medium supplemented with norbiotin it repressed synthesis of the enzyme. l-Arginine had no detectable effect on ornithine carbamoyltransferase synthesis by the yeast grown in biotin-deficient medium with or without norbiotin. l-Aspartate repressed synthesis of ornithine carbamoyltransferase in biotin-deficient yeast and completely nullified the stimulatory effect of norbiotin on synthesis of the enzyme in this yeast. 4. There was no increase in ornithine carbamoyltransferase synthesis in biotin-deficient yeast incubated in phosphate buffer, pH4.5, containing glucose and biotin or norbiotin. In biotin-deficient yeast suspended in complete medium containing an optimum concentration of biotin, there was an increase in ornithine carbamoyltransferase synthesis only after the onset of growth.
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PMID:A specific requirement for biotin in the synthesis of ornithine carbamoyltransferase by yeast. 596 54

In a previous study on yeast invertase (Reddy, A., and Maley, F. (1990) J. Biol. Chem. 265, 10817-10820), we identified Asp-23 through the procedures of affinity labeling and site-directed mutagenesis as a catalytic nucleophile. In the present study we undertook to determine other residues involved in the catalytic process. Earlier studies suggested histidine as a potential proton donor in the hydrolysis of sucrose, but by mutagenizing each of the enzyme's four histidines this amino acid was eliminated from consideration. Another candidate appeared to be cysteine, since iodine at about a 2-fold molar excess inactivated invertase by modifying both of the enzyme's cysteine residues. Dithiothreitol treatment restored the sulfhydryl groups and enzyme activity. Replacement of each of the cysteines with alanines revealed that C108A invertase retained full activity whereas C205A was reduced about 4-fold in its kcat. A comparison of the amino acid sequences of fructosylhydrolases revealed a conserved region coincident with Glu-204/Cys-205. Mutagenizing Glu-204 to Ala resulted in a 3, 000-fold reduction in the kcat of invertase indicating that Glu-204 plays a major role in catalysis. Based on these findings, a mechanism is proposed for the hydrolysis of sucrose which involves Asp-23 as a nucleophile and Glu-204 as an acid/base catalyst.
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PMID:Studies on identifying the catalytic role of Glu-204 in the active site of yeast invertase. 866 46

We constructed a system for the expression and secretion of mature hen lysozyme by yeast using an intermediate "secretion-signal cassette" vector, pKP1700, containing the yeast invertase signal sequence and an expression vector, pAM82, for secretion and maturation of the enzyme. Using this system, mutants of hen lysozyme were produced and the catalytic mechanism in hen lysozyme was definitely confirmed. The hydrolytic activity of D52A as to substrate (NAG)6 at pH 5.0 was obviously decreased to one-four hundredth of that of the wild type. The acidic limb of the pH-activity profile observed for the wild-type was not observed for D52A, and the pKa of Glu 35 on the alkaline limb was seen for both enzymes. Moreover, no structural change was detected on X-ray analysis of D52A. Therefore, we confirmed that dissociated Asp 52 assists catalysis by producing an electrostatic field and by stabilizing the oxocarbonium ion intermediate in the dissociated form.
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PMID:A mutation study of catalytic residue Asp 52 in hen egg lysozyme. 890 88

Streptococcus mutans glucosyltransferases (GTFs; GtfB, -C, and -D) synthesize water-soluble and -insoluble glucan polymers from sucrose. We have identified previously a conserved region of 19 amino acids (aa) (Gtf-P1; aa 409 to 427 of GtfB and aa 435 to 453 of GtfC) which is functionally important for both enzymatic activity and bacterial adherence. Monoclonal antibodies directed against Gtf-P1 selectively inhibited insoluble glucan synthesis by GtfB and -C but had no effect on soluble glucan synthesis by GtfD, suggesting that despite an apparent near identity of sequence, corresponding residues may function differently in these enzymes. To test this hypothesis, we used different strategies of mutagenesis to analyze amino acid residues of GtfB and GtfC in Gtf-P1. In-frame insertion of 6 amino acids preceding, or deletion of 14 amino acids within, this conserved region abolished the enzymatic activities of both GtfB and GtfC. Substitution of several residues in combination by random mutagenesis resulted in GtfB, but not GtfC, enzymes exhibiting decreased glucan synthesis and reduced rates of sucrose hydrolysis. Amino acid substitutions of Asp residues in GtfB or GtfC were found to be more critical for enzymatic activity than at other positions of this region. Interestingly, single mutation at Asp411 or Asp413 of GtfB resulted in enzymes retaining about 20% of wild-type activity, whereas mutagenesis of the corresponding Asp at position 437 or 439 in GtfC resulted in complete loss of enzymatic activity. Furthermore, single amino acid substitution of a Val residue between the two Asp residues enhanced the sucrase- and glucan-synthesizing activities of GtfB and GtfC. These results confirmed the report from another laboratory that Asp residues in the Gtf-P1 region are essential for enzymatic catalysis and provide new evidence that identical residues may function differently in closely related Gtf enzymes.
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PMID:Functional analyses of a conserved region in glucosyltransferases of Streptococcus mutans. 974 81

The extent of N-glycosylation of yeast external invertase at each of the 14 potential sites was determined by the combination of proteolytic digestions and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF-MS). The average molecular mass of the intact external invertase was determined as 97 kDa by MALDI/TOF-MS. The intact protein was digested with trypsin, Lys-C and Asp-N, followed by high-performance liquid chromatographic separation. The proteolytic digests were analyzed by MALDI/MS screening for the glycopeptides. The glycopeptides were then treated with peptide:N-glycosidase F (PNGase F) and/or endo-beta-N-acetylglucosaminidase (Endo H) and the molecular mass of the deglycosylated peptide was determined by MALDI/MS and matched with the peptide predicted by a computer program. The sequences of some peptides or deglycosylated peptides were identified by the MALDI post-source decay technique. The size of the oligosaccharide, the degree of glycosylation and the distribution of the oligosaccharides at each individual potential glycosylation site were characterized. This information goes for beyond previously published data and sometimes differs from them. During this study, the amino acid sequence originally derived from the DNA sequence of the gene coding for invertase was also verified and it was found that this protein when expressed from SUC2 gene might be created as more than one sequence which differ by a few amino acid substitutions (Asn58<-->Thr, Asn65-->His and Val412<-->Ala).
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PMID:Determination of N-linked glycosylation of yeast external invertase by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. 1022 60


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