Tryptophan phosphorescence study of enzyme flexibility and unfolding in laboratory-evolved thermostable esterases.
Directed evolution of p-nitrobenzyl esterase (pNB E) has yielded eight generations of increasingly thermostable variants. The most stable esterase, 8G8, has 13 amino acid substitutions, a melting temperature 17 degrees C higher than the wild-type enzyme, and increased hydrolytic activity toward p-nitrophenyl acetate (pNPA), the substrate used for evolution, at all temperatures. Room-temperature activities of the evolved thermostable variants range from 3.5 times greater to 4.0 times less than wild type. The relationships between enzyme stability, catalytic activity, and flexibility for the esterases were investigated using tryptophan phosphorescence. We observed no correlation between catalytic activity and enzyme flexibility in the vicinity of the tryptophan (Trp) residues. Increases in stability, however, are often accompanied by decreases in flexibility, as measured by Trp phosphorescence. Phosphorescence data also suggest that the N- and C-terminal regions of pNB E unfold independently. The N-terminal region appears more thermolabile, yet most of the thermostabilizing mutations are located in the C-terminal region. Mutational studies show that the effects of the N-terminal mutations depend on one or more mutations in the C-terminal region. Thus, the pNB E mutants are stabilized by long-range, cooperative interactions between distant parts of the enzyme.
Gershenson
A
A
Division of Chemistry & Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA.
Schauerte
J A
JA
Giver
L
L
Arnold
F H
FH
D019020:Directed Molecular Evolution; D004228:Dithionitrobenzoic Acid / Q000378:metabolism; D004795:Enzyme Stability; D004950:Esterases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008163:Luminescent Measurements; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D009596:Nitrophenols / Q000378:metabolism; D018583:Pliability; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013696:Temperature; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism*; D014732:Vibration
D009596:Nitrophenols; C008642:4-nitrophenyl acetate; D014364:Tryptophan; D004228:Dithionitrobenzoic Acid; D004950:Esterases