Differential effects of isomeric incorporation of fluorophenylalanines into PvuII endonuclease

Citation
Ma. Dominguez et al., Differential effects of isomeric incorporation of fluorophenylalanines into PvuII endonuclease, PROTEINS, 45(1), 2001, pp. 55-61
Citations number
37
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biochemistry & Biophysics
Journal title
PROTEINS-STRUCTURE FUNCTION AND GENETICS
ISSN journal
0887-3585 → ACNP
Volume
45
Issue
1
Year of publication
2001
Pages
55 - 61
Database
ISI
SICI code
0887-3585(20011001)45:1<55:DEOIIO>2.0.ZU;2-3
Abstract
Incorporation of fluorine into proteins has long served as a means of probi ng structure and function, yet there are few studies that examine the impac t of fluorine substitution, particularly at locations distant from the acti ve sites of enzymes. The flexibility of isomeric fluorine incorporation at Phe is used to explore subtle substitution effects on enzyme activity and c onformation. The unnatural amino acids o-, m-, and p-fluorophenylalanines w ere incorporated biosynthetically into the representative PvuII restriction endonuclease. Interestingly, m-fluoro-Phe-PvuII endonuclease exhibits very similar conformational stability to that of the native enzyme, but it exhi bits a reproducible, 2-fold higher average specific activity. Given the lev el of incorporation and the distribution of species, the species of modifie d enzyme responsible for this increase in specific activity is most likely even faster. Further, moving the fluorine atom from the meta- to the para-p osition of Phe results in a 4-fold decrease in specific activity and a decr ease in conformational stability of 1.5 kcal/mol. Since none of the Phe res idues in PvuII endonuclease lies near the DNA recognition or catalytic site s, this differential behavior alludes to the impact of subtle changes in en zyme conformation on endonuclease activity and suggests novel ways to influ ence catalytic behavior. (C) 2001 Wiley-Liss, Inc.