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Tryptophan Phosphorescence Study of Enzyme Flexibility and Unfolding in Laboratory-Evolved Thermostable Esterases
 

Summary: Tryptophan Phosphorescence Study of Enzyme Flexibility and Unfolding in
Laboratory-Evolved Thermostable Esterases
Anne Gershenson, Joseph A. Schauerte, Lori Giver,,| and Frances H. Arnold*,
DiVision of Chemistry & Chemical Engineering, California Institute of Technology, Pasadena, California 91125, and
Institute of Gerontology, UniVersity of Michigan, Ann Arbor, Michigan 48109
ReceiVed October 25, 1999; ReVised Manuscript ReceiVed February 11, 2000
ABSTRACT: 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 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.

  

Source: Arnold, Frances H. - Division of Chemistry and Chemical Engineering, California Institute of Technology

 

Collections: Chemistry; Biology and Medicine