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.
Department of Enzymology, Merck Research Laboratories, Rahway, New Jersey 07065.
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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