Design of an effective mechanism-based inactivator for a zinc protease.
AUTOR(ES)
Mobashery, S
RESUMO
(R)-2-Benzyl-5-cyano-4-oxopentanoic acid (compound 4) was studied as a mechanism-based inactivator (suicide substrate) for the zinc protease carboxypeptidase A (CPA; peptidyl-L-amino-acid hydrolase, EC 3.4.17.1). This compound was designed rationally based on the knowledge of the active site topology and the reported stereospecific proton exchange on ketonic substrate analogue (R)-3-(p-methoxybenzoyl)-2-benzylpropanoic acid [Sugimoto, T. & Kaiser, E. T. (1978) J. Am. Chem. Soc. 100, 7750-7751] by CPA. It is suggested that enzymic deprotonation on the C-5 methylene moiety may result in the transient formation of a ketenimine as the key intermediate that partitions between turnover and enzyme inactivation. The enzyme inactivation exhibited pseudo-first-order kinetics, was irreversible, and could be fully prevented in the presence of the reversible inhibitor benzyl-succinate. The inactivation rate constant, kintact, was evaluated to be 0.083 +/- 0.003 min-1 and kcat was measured at 1.78 +/- 0.06 min-1. In turn, a partition ratio of 28 +/- 3 was calculated. The reversible inhibitor constant (Ki) was measured at 1.8 +/- 0.5 microM, indicative of a high affinity for compound 4 shown by CPA; however, Km for the turnover process was determined at 4.93 +/- 0.43 mM. Kinetic analysis and labeling by the radioactive form of the inactivator suggested that the stoichiometry for protein modification by compound 4 approaches a 1:1 ratio.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=53308Documentos Relacionados
- Structure-assisted design of mechanism-based irreversible inhibitors of human rhinovirus 3C protease with potent antiviral activity against multiple rhinovirus serotypes
- Mechanism-Based Inactivation of Ammonia Monooxygenase in Nitrosomonas europaea by Allylsulfide
- Leukotriene A4 hydrolase: mapping of a henicosapeptide involved in mechanism-based inactivation.
- tert-Butylphenylacetylene Is a Potent Mechanism-Based Inactivator of Cytochrome P450 2B4: Inhibition of Cytochrome P450 Catalysis by Steric Hindrance
- Two-metal ion mechanism of RNA cleavage by HIV RNase H and mechanism-based design of selective HIV RNase H inhibitors