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| Predicate | Object |
|---|---|
| rdf:type | |
| lifeskim:mentions | |
| pubmed:dateCreated |
1992-3-31
|
| pubmed:abstractText |
1. The ratio of ebgA-gene product of ebgC-gene product in the functional aggregate of ebg beta-galactosidases was determined to be 1:1 by isolation of the enzyme from bacteria grown on uniformly radiolabelled amino acids and separation of the subunits by gel-permeation chromatography under denaturing conditions. 2. This datum, taken together with a recalculation of the previous ultracentrifuge data [Hall (1976) J. Mol. Biol. 107, 71-84], analytical gel-permeation chromatography and electron microscopy, strongly suggests an alpha 4 beta 4 quaternary structure for the enzyme. 3. The second chemical step in the enzyme turnover sequence, hydrolysis of the galactosyl-enzyme intermediate, is markedly slower for ebgab, having both Asp-97----Asn and Trp-977----Cys changes in the large subunit, than for ebga (having only the first change) and ebgb (having only the second), and is so slow as to be rate-determining even for an S-glycoside, beta-D-galactopyranosyl thiopicrate, as is shown by nucleophilic competition with methanol. 4. The selectivity of galactosyl-ebgab between water and methanol on a molar basis is 57, similar to the value for galactosyl-ebgb. 5. The equilibrium constant for the hydrolysis of lactose at 37 degrees C is 152 +/- 19 M, that for hydrolysis of allolactose is approx. 44 M and that for hydrolysis of lactulose is approx. 40 M. 6. A comparison of the free-energy profiles for the hydrolyses of lactose catalysed by the double mutant with those for the wild-type and the single mutants reveals that free-energy changes from the two mutations are not in general independently additive, but that the changes generally are in the direction predicted by the theory of Burbaum, Raines, Albery & Knowles [(1989) Biochemistry 28, 9283-9305] for an enzyme catalysing a thermodynamically irreversible reaction. 7. Michaelis-Menten parameters for the hydrolysis of six beta-D-galactopyranosylpyridinium ions and ten aryl beta-galactosides by ebgab were measured. 8. The derived beta 1g values are the same as those for ebgb (which has only the Trp-977----Cys change) and significantly different from those for ebgo (the wild-type enzyme) and ebga. 9. The alpha- and beta-deuterium secondary isotope effects on the hydrolysis of the galactosyl-enzyme of 1.08 and 1.00 are difficult to reconcile with the pyranose ring in this intermediate being in the 4C1 conformation.
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| pubmed:grant | |
| pubmed:commentsCorrections |
http://linkedlifedata.com/resource/pubmed/commentcorrection/1540130-105722,
http://linkedlifedata.com/resource/pubmed/commentcorrection/1540130-1107144,
http://linkedlifedata.com/resource/pubmed/commentcorrection/1540130-13979278,
http://linkedlifedata.com/resource/pubmed/commentcorrection/1540130-14155091,
http://linkedlifedata.com/resource/pubmed/commentcorrection/1540130-14188863,
http://linkedlifedata.com/resource/pubmed/commentcorrection/1540130-2085322,
http://linkedlifedata.com/resource/pubmed/commentcorrection/1540130-2271534,
http://linkedlifedata.com/resource/pubmed/commentcorrection/1540130-2271539,
http://linkedlifedata.com/resource/pubmed/commentcorrection/1540130-2498341,
http://linkedlifedata.com/resource/pubmed/commentcorrection/1540130-2505746,
http://linkedlifedata.com/resource/pubmed/commentcorrection/1540130-2513251,
http://linkedlifedata.com/resource/pubmed/commentcorrection/1540130-2515108,
http://linkedlifedata.com/resource/pubmed/commentcorrection/1540130-2611230,
http://linkedlifedata.com/resource/pubmed/commentcorrection/1540130-2806240,
http://linkedlifedata.com/resource/pubmed/commentcorrection/1540130-3083779,
http://linkedlifedata.com/resource/pubmed/commentcorrection/1540130-323855,
http://linkedlifedata.com/resource/pubmed/commentcorrection/1540130-3939707,
http://linkedlifedata.com/resource/pubmed/commentcorrection/1540130-3939708,
http://linkedlifedata.com/resource/pubmed/commentcorrection/1540130-4124306,
http://linkedlifedata.com/resource/pubmed/commentcorrection/1540130-4575974,
http://linkedlifedata.com/resource/pubmed/commentcorrection/1540130-4598756,
http://linkedlifedata.com/resource/pubmed/commentcorrection/1540130-4868117,
http://linkedlifedata.com/resource/pubmed/commentcorrection/1540130-6254948,
http://linkedlifedata.com/resource/pubmed/commentcorrection/1540130-6411710,
http://linkedlifedata.com/resource/pubmed/commentcorrection/1540130-6793063,
http://linkedlifedata.com/resource/pubmed/commentcorrection/1540130-6801019,
http://linkedlifedata.com/resource/pubmed/commentcorrection/1540130-794482,
http://linkedlifedata.com/resource/pubmed/commentcorrection/1540130-999839
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| pubmed:language |
eng
|
| pubmed:journal | |
| pubmed:citationSubset |
IM
|
| pubmed:chemical | |
| pubmed:status |
MEDLINE
|
| pubmed:month |
Feb
|
| pubmed:issn |
0264-6021
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| pubmed:author | |
| pubmed:issnType |
Print
|
| pubmed:day |
15
|
| pubmed:volume |
282 ( Pt 1)
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| pubmed:geneSymbol |
ebg,
ebg<up>ab</up>,
ebgA,
ebgC
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| pubmed:owner |
NLM
|
| pubmed:authorsComplete |
Y
|
| pubmed:pagination |
155-64
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| pubmed:dateRevised |
2010-11-18
|
| pubmed:meshHeading |
pubmed-meshheading:1540130-Biological Evolution,
pubmed-meshheading:1540130-Escherichia coli,
pubmed-meshheading:1540130-Genes, Bacterial,
pubmed-meshheading:1540130-Kinetics,
pubmed-meshheading:1540130-Lactose,
pubmed-meshheading:1540130-Macromolecular Substances,
pubmed-meshheading:1540130-Mathematics,
pubmed-meshheading:1540130-Models, Genetic,
pubmed-meshheading:1540130-Substrate Specificity,
pubmed-meshheading:1540130-Thermodynamics,
pubmed-meshheading:1540130-beta-Galactosidase
|
| pubmed:year |
1992
|
| pubmed:articleTitle |
The catalytic consequences of experimental evolution. Studies on the subunit structure of the second (ebg) beta-galactosidase of Escherichia coli, and on catalysis by ebgab, an experimental evolvant containing two amino acid substitutions.
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| pubmed:affiliation |
Department of Organic Chemistry, University of Bristol, U.K.
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| pubmed:publicationType |
Journal Article,
Research Support, U.S. Gov't, P.H.S.,
Research Support, Non-U.S. Gov't
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