Properties of Trypsin-Mediated Activation of Aspartase from Hafnia alvei

  • Lee, Min-Sub (Department of Chemistry, Hanyang University) ;
  • Choi, Kyoung-Jae (Department of Chemistry, Hanyang University) ;
  • Kwom, Si-Joong (Department of Chemistry, Hanyang University) ;
  • Kang, In-Sug (Department of Molecular Biology, College of Medicine, Kyung Hee University) ;
  • Ha, Joo-Hun (Department of Molecular Biology, College of Medicine, Kyung Hee University) ;
  • Kim, Sung-Soo (Department of Molecular Biology, College of Medicine, Kyung Hee University) ;
  • Han, Myung-Soo ;
  • Yoon, Moon-Young (Department of Chemistry, Hanyang University)
  • Received : 0
  • Accepted : 0
  • Published : 0

Abstract

Treatment of Hafnia alvei aspartase with limited tryptic digestion resulted in a marked increase in enzymatic activity. The activation required a few minutes to attain maximum level and, thereafter, the activity gradually decreased to complete inactivation. The degree of cleavage associated with the activation was extremely small as judged by SDS-PAGE. Upon activation, the optimum pH and temperature were essentially unchanged. When trypsin-activated enzyme was denatured in 4 M guanidine-HCI followed by removal of the denaturant by dilution, the restoration of activity was similar (40%) to that of the native enzyme, indicating a degree of stability. The $pK_a$ obtained on the acidic side and the $pK_b$ obtained on the basic side of trypsin-activated aspartase were 6.6 and 8.6, respectively, the same as those of the native aspartase, indicating that aspartase may exist in a stable conformation after limited tryptic digestion. These results indicate that the activation of H. alvei may be mediated by a conformational change away from the active site of individual subunits.

Keywords

Aspartase;Trypsin activation;Hafnia alvei