Enzyme Assays Aminopeptidase Activity

Application and Principle This procedure is used to determine leucine aminopeptidase activity in enzyme preparations derived from Lactococcus lactis. The assay is based on the rate of absorbance change over 5 min at 30° the change in absorbance is due to liberatedp-nitroaniline from the hydrolysis of leucine p-nitroanilide. 

Aspartate a-decarboxylase 753, 755 Aspartate p-decarboxylase 746 Aspartate racemase 741 Aspartic acid (Asp, D) 52, 53s biosynthesis 517 pXa value of 293, 487 Aspartic proteases 621-625 Aspartyl aminopeptidase 621 p-Aspartyl phosphate 539, 540s Assays of enzyme activity 456 Assembly core of virus shell 365 Assembly pathway... 

The /V-formylmethionine of a nascent protein synthesized in bacteria is removed by the sequential activities of PDF and a methionine aminopeptidase to generate the mature protein. The gene encoding PDF was cloned and overexpressed in E. coli by Meinnel and coworkers (1993). The PDF enzyme has an unusual metal ion (Fe2+) as its catalyst. However, the ferrous ion in this enzyme is unstable and can be quickly and irreversibly oxidized to ferric ion, rapidly inactivating the enzyme. PDF-based assay development therefore depended on the ability of nickel ion to replace ferrous ion in vitro, increasing the stability of the enzyme and maintaining its enzymatic activity (Groche et al., 1998 Clements et al., 2001 Hackbarth et al., 2002). 

In related work, Dong and Martin developed an assay that detects the catalytic activity of the enzymes pig liver esterase and porcine kidney leucine aminopeptidase by using substrates which have been labeled with metal-binding ligands [57], The enzymes catalyze changes in the substrates that affect their ability to bind to non-luminescent Ru complexes to form mixed-ligand complexes capable of ECL. 

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