Substrate specificities of the peptidyl prolyl cis-trans isomerase activities of cyclophilin and FK-506 binding protein: evidence for the existence of a family of distinct enzymes.
Substrate specificities, as reflected in kc/Km, were determined for the peptidyl prolyl cis-trans isomerase activities of cyclophilin and the FK-506 binding protein (FKBP). The substrates investigated were peptides of the general structure Suc-Ala-Xaa-Pro-Phe-p-nitroanilide, where Xaa = Gly, Ala, Val, Leu, Phe, His, Lys, on Glu. While kc/Km for cyclophilin-catalyzed isomerization shows little dependence on Xaa, kc/Km values for FKBP-catalyzed isomerization display a marked dependence on Xaa and vary over 3 orders of magnitude. An important outcome of this work is the discovery that Suc-Ala-Leu-Pro-Phe-pNA is a reactive substrate for FKBP (kc/Km = 640,000 M-1 s-1). This substrate can be used with FKBP concentrations that are low enough to allow, for the first time, accurate determinations of Ki values for tight-binding inhibitors of FKBP. Using this new assay, we found that FK-506 inhibits FKBP with Ki = 1.7 +/- 0.6 nM. The results of this work support the hypothesis that cyclophilin and FKBP are members of a family of peptidyl prolyl cis-trans isomerases and that the members of this family possess distinct substrate specificities that allow them to play diverse physiologic roles.
Harrison
R K
RK
Department of Enzymology, Merck Research Laboratories, Rahway, New Jersey 07065.
Stein
R L
RL
D000591:Amino Acid Isomerases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000900:Anti-Bacterial Agents / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D002384:Catalysis; D003524:Cyclosporins / Q000493:pharmacokinetics; D007166:Immunosuppressive Agents / Q000493:pharmacokinetics*; D007700:Kinetics; D019696:Peptidylprolyl Isomerase; D013379:Substrate Specificity; D016559:Tacrolimus; D013816:Thermodynamics
D000900:Anti-Bacterial Agents; D002352:Carrier Proteins; D003524:Cyclosporins; D007166:Immunosuppressive Agents; D000591:Amino Acid Isomerases; D019696:Peptidylprolyl Isomerase; D016559:Tacrolimus