Controlling the enzymatic activity of a restriction enzyme by light.
For many applications it would be desirable to be able to control the activity of proteins by using an external signal. In the present study, we have explored the possibility of modulating the activity of a restriction enzyme with light. By cross-linking two suitably located cysteine residues with a bifunctional azobenzene derivative, which can adopt a cis- or trans-configuration when illuminated by UV or blue light, respectively, enzymatic activity can be controlled in a reversible manner. To determine which residues when cross-linked show the largest "photoswitch effect," i.e., difference in activity when illuminated with UV vs. blue light, > 30 variants of a single-chain version of the restriction endonuclease PvuII were produced, modified with azobenzene, and tested for DNA cleavage activity. In general, introducing single cross-links in the enzyme leads to only small effects, whereas with multiple cross-links and additional mutations larger effects are observed. Some of the modified variants, which carry the cross-links close to the catalytic center, can be modulated in their DNA cleavage activity by a factor of up to 16 by illumination with UV (azobenzene in cis) and blue light (azobenzene in trans), respectively. The change in activity is achieved in seconds, is fully reversible, and, in the case analyzed, is due to a change in V(max) rather than K(m).
Schierling
Benno
B
Institute of Biochemistry, Justus-Liebig University, Giessen, Germany.
Noël
Ann-Josée
AJ
Wende
Wolfgang
W
Hien
Le Thi
le T
Volkov
Eugeny
E
Kubareva
Elena
E
Oretskaya
Tatiana
T
Kokkinidis
Michael
M
Römpp
Andreas
A
Spengler
Bernhard
B
Pingoud
Alfred
A
D017398:Alternative Splicing; D001391:Azo Compounds / Q000737:chemistry; D001665:Binding Sites; D055162:Biocatalysis; D003432:Cross-Linking Reagents / Q000737:chemistry; D018360:Crystallography, X-Ray; D004262:DNA Restriction Enzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000528:radiation effects; D007536:Isomerism; D008027:Light; D008958:Models, Molecular; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D017434:Protein Structure, Tertiary; D014466:Ultraviolet Rays
D001391:Azo Compounds; D003432:Cross-Linking Reagents; D004262:DNA Restriction Enzymes; C009850:azobenzene