Linked Life Data

10438473

Source:http://linkedlifedata.com/resource/pubmed/id/10438473

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
33
pubmed:dateCreated
1999-9-1
pubmed:abstractText
We investigated the effects of mutations at positions 164 and 179 of the TEM(pUC19) beta-lactamase on turnover of substrates. The direct consequence of some mutations at these sites is that clinically important expanded-spectrum beta-lactams, such as third-generation cephalosporins, which are normally exceedingly poor substrates for class A beta-lactamases, bind the active site of these mutant enzymes more favorably. We employed site-saturation mutagenesis at both positions 164 and 179 to identify mutant variants of the parental enzyme that conferred resistance to expanded-spectrum beta-lactams by their enhanced ability to turn over these antibiotic substrates. Four of these mutant variants, Arg(164) --> Asn, Arg(164) --> Ser, Asp(179) --> Asn, and Asp(179) --> Gly, were purified and the details of their catalytic properties were examined in a series of biochemical and kinetic experiments. The effects on the kinetic parameters were such that either activity with the expanded-spectrum beta-lactams remained unchanged or, in some cases, the activity was enhanced. The affinity of the enzyme for these poorer substrates (as defined by the dissociation constant, K(s)) invariably increased. Computation of the microscopic rate constants (k(2) and k(3)) for turnover of these poorer substrates indicated either that the rate-limiting step in turnover was the deacylation step (governed by k(3)) or that neither the acylation nor deacylation became the sole rate-limiting step. In a few instances, the rate constants for both the acylation (k(2)) and deacylation (k(3)) of the extended-spectrum beta-lactamase were enhanced. These results were investigated further by molecular modeling experiments, using the crystal structure of the TEM(pUC19) beta-lactamase. Our results indicated that severe steric interactions between the large 7beta functionalities of the expanded-spectrum beta-lactams and the Omega-loop secondary structural element near the active site were at the root of the low affinity by the enzyme for these substrates. These conclusions were consistent with the proposal that the aforementioned mutations would enlarge the active site, and hence improve affinity.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Aug
pubmed:issn
0021-9258
pubmed:author
pubmed:issnType
Print
pubmed:day
13
pubmed:volume
274
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
23052-60
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
1999
pubmed:articleTitle
Effects on substrate profile by mutational substitutions at positions 164 and 179 of the class A TEM(pUC19) beta-lactamase from Escherichia coli.
pubmed:affiliation
Department of Medicine, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, Non-U.S. Gov't