Evidence for the negative cooperativity of the two active sites within bovine somatic angiotensin‐converting enzyme

FEBS Letters

Kost, Olga A²  Pozdnev, Vladimir F¹  Binevski, Peter V²  Sizova, Elena A² 

[1] Institute of Biomedical Chemistry, Russian Academy of Medical Sciences, ul. Pogodinskaya 10, Moscow 119832, Russia;Chemistry Faculty, Lomonosov Moscow State University, Moscow 119992, Russia

关键词: Angiotensin-converting enzyme;    N-domain;    C-domain;    Negative cooperativity;    ACE;    angiotensin-converting enzyme;    FA-Phe-Gly-Gly;    N-(3-[2-furyl]acryloyl)-L-phenylalanyl-glycyl-glycine;    FA-Phe-Ala-Ala;    N-(3-[2-furyl]acryloyl)-L-phenylalanyl-L-alanyl-L-alanine;    FA-Phe-Ala-Lys;    N-(3-[2-furyl]acryloyl)-L-phenylalanyl-L-alanyl-L-lysine;    FA-Phe-Ala-Pro;    N-(3-[2-furyl]acryloyl)-L-phenylalanyl-L-alanyl-L-proline;    FA-Phe-Phe-Arg;    N-(3-[2-furyl]acryloyl)-L-phenylalanyl-L-phenylalanyl-L-arginine;    Hip-His-Leu;    N-benzoyl-glycyl-L-histidyl-L-leucine;    Cbz-Phe-His-Leu;    N-carbobenzoxy-L-phenylalanyl-L-histidyl-L-leucine;    captopril;    (2S)-1-(3-mercapto-2-methylpropionyl)-L-proline;    lisinopril;    (S)-N α-(1-carboxy-3-phenylpropyl)-L-lysyl-L-proline;   

DOI  :  10.1016/S0014-5793(03)00825-1

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

The somatic isoform of angiotensin-converting enzyme (ACE) consists of two homologous domains (N- and C-domains), each bearing a catalytic site. We have used the two-domain ACE form and its individual domains to compare characteristics of different domains and to probe mutual functioning of the two active sites within a bovine ACE molecule. The substrate Cbz-Phe-His-Leu (N-carbobenzoxy-L-phenylalanyl-L-histidyl-L-leucine; from the panel of seven) was hydrolyzed faster by the N-domain, the substrates FA-Phe-Gly-Gly (N-(3-[2-furyl]acryloyl)-L-phenylalanyl-glycyl-glycine) and Hip-His-Leu (N-benzoyl-glycyl-L-histidyl-L-leucine) were hydrolyzed by both domains with equal rates, while other substrates were preferentially hydrolyzed by the C-domain. The inhibitor captopril ((2S)-1-(3-mercapto-2-methylpropionyl)-L-proline) bound to the N-domain more effectively than to the C-domain, whereas lisinopril ((S)-N ^α-(1-carboxy-3-phenylpropyl)-L-lysyl-L-proline) bound to equal extent with all ACE forms. However, active site titration with lisinopril assayed by hydrolysis of FA-Phe-Gly-Gly revealed that 1 mol of inhibitor/mol of enzyme abolished the activity of either two-domain or single-domain ACE forms, indicating that a single active site functions in bovine somatic ACE. Neither of the k _cat values obtained for somatic enzyme was the sum of k _cat values for individual domains, but in every case the value of the catalytic constant of the hydrolysis of the substrate by the two-domain ACE represented the mean quantity of the values of the corresponding catalytic constants obtained for single-domain forms. The results indicate that the two active sites within bovine somatic ACE exhibit strong negative cooperativity.

【 授权许可】

Unknown