The Slow and Tight Binding of MR-387A to Aminopeptidase N

  • CHUNG, MYUNG-CHUL (Enzyme Inhibitor Research Unit, Korea Research Institute of Bioscience and Biotechnology, KIST) ;
  • HYO-KON CHUN (Enzyme Inhibitor Research Unit, Korea Research Institute of Bioscience and Biotechnology, KIST) ;
  • HO-JAE LEE (Enzyme Inhibitor Research Unit, Korea Research Institute of Bioscience and Biotechnology, KIST) ;
  • CHOONG-HWAN LEE (Enzyme Inhibitor Research Unit, Korea Research Institute of Bioscience and Biotechnology, KIST) ;
  • SU-IL KIM (Department of Agricultural Chemistry, Seoul National University) ;
  • YUNG-HEE KHO (Enzyme Inhibitor Research Unit, Korea Research Institute of Bioscience and Biotechnology, KIST)
  • Published : 1996.08.01

Abstract

MR-387A [(2S, 3R)-2-hydroxy-3-amino-4-phenylbutanoyl-L-valyl-L-prolyl-(2, 4-trans)- L-4-hydroxy-proline] reversibly inhibits aminopeptidase N (BC 3.4.11.2) in a process that is remarkable for its unusual degree of time dependence. The time required to inactivate the enzyme by 50$%$ ($t_{1/2}$) for establishing steady-state levels of $EI^*$complex was approximately 5 minutes. This indicates that the inhibition is a slow-binding process. In dissociation experiments of $EI^*$ complex, enzymic activity was regained slowly in a quadratic equation, indicating that the inhibition of aminopeptidase N by MR-387A is tight-binding and reversible. Thus, the binding of MR-387A by aminopeptidase N is slow and tight, with $K_{i}$ (for initial collision complex, EI) and $K_i{^*}$ (for final tightened complex, $EI^*$) of $2.2\times10^{-8}$ M (from Lineweaver-Burk plot) and $4.4\times10^{-10}$ M (from rate constants), respectively. These data indicate that MR-387A and aminopeptidase N are bound approximately 200-fold more tightly in the final $EI^*$complex than in the initial collision EI complex.