Active site probes of flavoproteins. Determination of the solvent accessibility of the flavin position 8 for a series of flavoproteins.
The chemical reactivity of 8-chloroflavins and 8-mercaptoflavins has been exploited in order to examine the orientation of protein-bound flavins relative to solvent. The apoprotein form of a series of flavoproteins was prepared and the native flavin was replaced by either 8-Cl-flavin or 8-mercaptoflavin (FAD, FMN, or riboflavin form as was appropriate). The reconstituted proteins were exposed to reagents capable of reacting with the group at position 8. The 8-Cl-proteins were challenged with sodium sulfide and thiophenol, while the 8-mercaptoproteins were faced with iodoacetamide and iodoacetic acid. The kinetics of the ensuing reactions served as a measure of the solvent availability of position 8 for the protein-bound flavin. These studies indicated that position 8 of flavin bound to melilotate hydroxylase, D-amino acid oxidase, old yellow enzyme, p-OH-benzoate hydroxylase, and flavodoxin is accessible to solvent, while position 8 on L-lactate oxidase, glucose oxidase, putrescine oxidase, and riboflavin-binding protein appears to be inaccessible. For luciferase, D-lactate dehydrogenase, and xanthine oxidase, the data suggest that position 8 is exposed but the results are inconclusive. The effect of ligand binding on the accessibility of position 8 was also studied. NADPH binding to 8-mercapto old yellow enzyme and benzoate binding to 8-Cl-D-amino acid oxidase results in complete blockage of previously available position 8. On the other hand, p-OH-benzoate hydroxylase and melilotate hydroxylase bind their respective substrates (p-OH-benzoate and melilotate) without significantly altering the reactivity of position 8.
D001059:Apoproteins; D001665:Binding Sites; D005486:Flavin Mononucleotide / Q000031:analogs & derivatives*; D005182:Flavin-Adenine Dinucleotide / Q000031:analogs & derivatives*; D005420:Flavoproteins; D008024:Ligands; D011485:Protein Binding; D012997:Solvents; D013329:Structure-Activity Relationship
D001059:Apoproteins; D005420:Flavoproteins; D008024:Ligands; D012997:Solvents; D005182:Flavin-Adenine Dinucleotide; C017123:8-chloro-FAD; C023405:8-chloro-FMN; D005486:Flavin Mononucleotide