Stability of TEM beta-lactamase mutants hydrolyzing third generation cephalosporins.
The stability properties of six natural mutants of the TEM-1 beta-lactamase have been studied. The glutamate to lysine substitution at positions 104 and 240 stabilize the enzyme. Conversely, the G238S mutant's decreased stability might reflect an altered conformation of the active site and thus be related to the modified substrate profile. The relative stability of the R164S and R164H mutants is explained by the formation of a hydrogen bond between these residues and Asp-179 conferring a somewhat different structure to the omega loop and thus also explaining the extended substrate profile of these mutants. The loss of stability of the R164H mutant with increasing pH values can be explained by the titration of a hydrogen bond between the N delta of His-164 and the O delta of Asp-179. The properties of the G238S + E104K double mutant which is the most active against third-generation cephalosporins result from a balance of destabilizing and stabilizing substitutions, and their effects seem to be additive. The behavior of the R164S + E240K mutant might be explained on the basis of a similar compensation phenomenon.
Raquet
X
X
Centre d'Ingénierie des Protéines, Université de Liège, Sart-Tilman, Belgium.
Vanhove
M
M
Lamotte-Brasseur
J
J
Goussard
S
S
Courvalin
P
P
Frère
J M
JM
D001120:Arginine; D018550:Cephalosporin Resistance; D002511:Cephalosporins / Q000378:metabolism*; D004795:Enzyme Stability / Q000235:genetics; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008956:Models, Chemical; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D012694:Serine; D013696:Temperature; D013816:Thermodynamics; D014357:Trypsin / Q000378:metabolism; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D002511:Cephalosporins; D012694:Serine; D006639:Histidine; D001120:Arginine; D014357:Trypsin; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1