Differential effects of isomeric incorporation of fluorophenylalanines into PvuII endonuclease.
Incorporation of fluorine into proteins has long served as a means of probing structure and function, yet there are few studies that examine the impact of fluorine substitution, particularly at locations distant from the active sites of enzymes. The flexibility of isomeric fluorine incorporation at Phe is used to explore subtle substitution effects on enzyme activity and conformation. The unnatural amino acids o-, m-, and p-fluorophenylalanines were 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 exhibits a reproducible, 2-fold higher average specific activity. Given the level of incorporation and the distribution of species, the species of modified enzyme responsible for this increase in specific activity is most likely even faster. Further, moving the fluorine atom from the meta- to the para-position of Phe results in a 4-fold decrease in specific activity and a decrease in conformational stability of 1.5 kcal/mol. Since none of the Phe residues in PvuII endonuclease lies near the DNA recognition or catalytic sites, this differential behavior alludes to the impact of subtle changes in enzyme conformation on endonuclease activity and suggests novel ways to influence catalytic behavior.
Dominguez
M A
MA
Department of Biochemistry & Biophysics, Texas A&M University College Station, Texas 77843-2128, USA.
Thornton
K C
KC
Melendez
M G
MG
Dupureur
C M
CM
D000495:Allosteric Site; D019943:Amino Acid Substitution; D001665:Binding Sites; D002384:Catalysis; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli; D005461:Fluorine / Q000378:metabolism*; D008958:Models, Molecular; D015335:Molecular Probes / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000378:metabolism*; D018583:Pliability; D011487:Protein Conformation; D015202:Protein Engineering; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013816:Thermodynamics
D015335:Molecular Probes; D005461:Fluorine; D010649:Phenylalanine; C025630:CAGCTG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific