The nature of chloride dependency on the activity of angiotensin I-converting enzyme which catalyses the release of C-terminal dipetide from angiotensin I decapeptide was studied by kinetic analysis using Hippuryl-Gly-Gly and N-Cbz-Gly-Gly-Pro as the substrate analogous.
The angiotensin I-converting enzyme acts as a general dipeptidyl carboxypeptidase, but unlike other peptidase it shows a strong dependency on the presence of chloride ion for the full activity. When the activity of the enzyme was measured by a modified ninhydrin method employing an ion-exchange column of Aminex A-4, a sigmoidal curve of the enzyme activity profile was obtained as a function of chloride concentration. No change in the enzyme activity was found with other halogen ions such as fluoride, bromide, or iodide. The enzyme seems to be very specific toward chloride ion as a cofactor for the activity.
When the enzyme activity was demonstrated by varying the concentration of the peptide substrate at different fixed concentrations of chloride ion, the resulting reciprocal plots showed a typical converging pattern indicating a possible involvement of a ternary complex between the enzyme, the peptide substrate and chloride ion.
From the results it is suggested that chloride may increase the rate of enzyme reaction by decreasing the apparent $K_m$ value. The calculated values for Hippuryl-Gly-Gly and N-Cbz-Gly-Gly-Pro were 0.07 and 0.46, respectively. Based on the kinetic data obtained, it is suggested that chloride may have a role as an allosteric effector in the binding of the enzyme and substrate.
Angiotensin I-converting enzyme 은 생체내에서 Angiotensin I 을 Angiotensin II 로 전환시키며, Bradykinin 을 불 활성화 시킴으로써 본태성 고혈압 및 신성 고혈압을 일으키는 중요한 인자로 간주되고 있다.
본 실험에서는 여러 종류의 artificial 기질을 사용하여 dipeptidyl carboxypeptidase 로서의 Angiotensin I-converting enzyme 의 특성을 연구하였다. 효소의 활성을 automated minhydrin method 를 사용하여 측정하였다.
C-terminal free COOH group 이 기질의 절대 요구성 임을 알 수 있었다. Angiotensin I-converting enzyme 의 활성은 small peptide 및 아미노산과 이의 유도체에 의하여 저하되지 않았다. Hippuryl-Gly-Gly, N-$Cb_3$-Gly-Gly-Pro 두 기질을 사용하여 효소의 활성에 영향을 미치는 chloride 의 기능을 연구하였다.
Angiotensin I-converting enzyme 의 활성은 chloride ion 에 의하여 activation 되며, chloride ion 은 non-essential activator 로서 E.Cl.S 의 ternary complex 를 형성함을 알수 있었다.
Chloride ion 의 생화학적 의미는 효소와 결합하여 기질의 친화력을 증가시켜 Angiotensin I-converting enzyme 의 활성을 activation 시키는 것으로 간주된다.