Document Detail


Threonine deaminase from a nonsense mutant of Escherichia coli requiring isoleucine or pyridoxine: evidence for half-of-the-sites reactivity.
MedLine Citation:
PMID:  770416     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
The mutant IP7 of Escherichia coli B requires isoleucine or pyridoxine for growth as a consequence of a mutation in the gene coding for biosynthetic threonine deaminase. The mutation of IP7 was shown to be of the nonsense type by the following data: (1) reversion to isoleucine prototrophy involves the formation of external suppression at a high frequency, as shown by transduction experiments; and (ii) the isoleucine requirement is suppressed by lysogenization with a phage carrying the amber suppressor su-3. Cell extracts of the mutant strain contain a low activity of threonine deaminase. The possibility that this activity is biodegradative was ruled out by kinetic experiments. The mutant threonine deaminase was purified to homogeneity by conventional procedures. The enzyme is a dimer of identical subunits of an approximate molecular weight of 43,000 (Grimminger and Feldner, 1974), whereas the wild-type enzyme is a tetramer of 50,000-dalton subunits (Calhoun et al., 1973; Grimminger et al., 1973). The mutant enzyme is not inhibited by isoleucine and does not bind isoleucine, as shown by equilibrium dialysis experiments. Pyridoxal phosphate enhances the maximum catalytic activity of the mutant enzyme by a factor of five, whereas the wild-type enzyme is not affected. In wild-type and mutant threonine deaminase the ratio of protein subunits and bound pyridoxal phosphate is 2:1. The activation of threonine deaminase from strain IP7 is due to a second coenzyme binding site, as shown by (i) spectrophotometric titration of the enzyme with pyridoxal phosphate and by (ii) measurement the pyridoxal phosphate content of the enzyme after sodium borohydride reduction of the protein. The observation of one pyridoxal phosphate binding site per peptide dimer in the wild-type enzyme and of two binding sites per dimer in the mutant strongly suggests that one of the potential sites in the wild-type enzyme is masked by allosteric effects. The factors responsible for the half-of-the-sites reactivity of the coenzyme sites appear to be nonoperative in the mutant protein.
Authors:
J Feldner; H Grimminger
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Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  Journal of bacteriology     Volume:  126     ISSN:  0021-9193     ISO Abbreviation:  J. Bacteriol.     Publication Date:  1976 Apr 
Date Detail:
Created Date:  1976-07-06     Completed Date:  1976-07-06     Revised Date:  2010-09-02    
Medline Journal Info:
Nlm Unique ID:  2985120R     Medline TA:  J Bacteriol     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  100-7     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
Binding Sites
Cell-Free System
Enzyme Activation
Escherichia coli / enzymology*,  metabolism
Hydro-Lyases* / biosynthesis
Isoleucine / metabolism*
Molecular Weight
Mutation*
Pyridoxal Phosphate / metabolism
Pyridoxine / metabolism*
Threonine Dehydratase* / biosynthesis,  metabolism
Chemical
Reg. No./Substance:
54-47-7/Pyridoxal Phosphate; 65-23-6/Pyridoxine; 73-32-5/Isoleucine; EC 4.2.1.-/Hydro-Lyases; EC 4.3.1.19/Threonine Dehydratase
Comments/Corrections

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