Kinetic characterization of ribonuclease S mutants containing photoisomerizable phenylazophenylalanine residues.
Incorporation of the photoisomerizable amino acid phenylazophenylalanine (PAP) into enzyme structures has been proposed as a strategy for photoswitching enzyme activity. To evaluate the strengths and limitations of this approach to enzyme photo-control, we performed a kinetic analysis of RNase S analogues containing PAP in positions 4, 7, 8, 10, 11 or 13. For an enzyme containing a single PAP group, the maximum extent of photoconversion (between approximately 96% trans/4% cis and 10% trans/90% cis under standard conditions) sets a limit on the maximum fold change in the initial rate of approximately 25-fold, if the cis form is the more active isomer, and approximately 10-fold if the trans form is more active. This extent of photoswitching was not realized in the present case because the effects of photoisomerization on kinetic constants were small and distributed among effects on S-peptide binding, substrate binding and the rate of the chemical step. These results suggest that photoisomerization could substantially alter enzyme kinetic constants but that a directed combinatorial approach might be required for realizing maximal photo-control in such systems. The limit set by the extent of photoconversion might be overcome by coupling multiple PAP groups to one enzyme or by altering the behaviour of a system that required oligomerization for activity.
James
D A
DA
Department of Chemistry, University of Toronto, 80 St George St., Toronto, Canada, M5S 3H6.
Burns
D C
DC
Woolley
G A
GA
D000595:Amino Acid Sequence; D001391:Azo Compounds / Q000737:chemistry; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007536:Isomerism; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000737:chemistry*; D010777:Photochemistry; D012260:Ribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
C087328:4-phenylazophenylalanine; D001391:Azo Compounds; D007527:Isoenzymes; D010649:Phenylalanine; D012260:Ribonucleases; C022542:ribonuclease S