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

Biochemistry. 1990 Apr 24;29(16):3813-6. doi: 10.1021/bi00468a001.

Abstract

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.

MeSH terms

  • Amino Acid Isomerases / antagonists & inhibitors
  • Amino Acid Isomerases / metabolism*
  • Anti-Bacterial Agents / metabolism*
  • Carrier Proteins / metabolism*
  • Catalysis
  • Cyclosporins / pharmacokinetics
  • Immunosuppressive Agents / pharmacokinetics*
  • Kinetics
  • Peptidylprolyl Isomerase
  • Substrate Specificity
  • Tacrolimus
  • Thermodynamics

Substances

  • Anti-Bacterial Agents
  • Carrier Proteins
  • Cyclosporins
  • Immunosuppressive Agents
  • Amino Acid Isomerases
  • Peptidylprolyl Isomerase
  • Tacrolimus