A new colorimetric assay for methionyl aminopeptidases: examination of the binding of a new class of pseudopeptide analog inhibitors.
A direct and convenient spectrophotometric assay has been developed for methionine aminopeptidases (MetAPs). The method employs the hydrolysis of a substrate that is a methionyl analogue of p-nitroaniline (L-Met-p-NA), which releases the chromogenic product p-nitroaniline. This chromogenic product can be monitored continuously using a UV-Vis spectrophotometer set at 405 nm. The assay was tested with the type I MetAP from Escherichia coli (EcMetAP-I) and the type II MetAP from Pyrococcus furiosus (PfMetAP-II). Using L-Met-p-NA, the kinetic constants k(cat) and K(m) were determined for EcMetAP-I and PfMetAP-II and were compared with those obtained with a "standard" high-performance liquid chromatography (HPLC) discontinuous assay. The assay has also been used to determine the temperature dependence of the kinetic constant k(cat) for PfMetAP-II as well as to screen two novel pseudopeptide inhibitors of MetAPs. The results demonstrate that L-Met-p-NA provides a fast, convenient, and effective substrate for both type I and type II MetAPs and that this substrate can be used to quickly screen inhibitors of MetAPs.
Mitra
Sanghamitra
S
Department of Chemistry and Biochemistry, Utah State University, Logan, 84322, USA.
Dygas-Holz
Anna M
AM
Jiracek
Jiri
J
Zertova
Miroslava
M
Zakova
Lenka
L
Holz
Richard C
RC
D000626:Aminopeptidases / Q000032:analysis* / Q000037:antagonists & inhibitors*; D003124:Colorimetry / Q000379:methods*; D004791:Enzyme Inhibitors / Q000145:classification* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D006868:Hydrolysis; D007700:Kinetics; D008715:Methionine / Q000031:analogs & derivatives / Q000378:metabolism; D063208:Methionyl Aminopeptidases; D010455:Peptides / Q000378:metabolism; D020027:Pyrococcus furiosus / Q000201:enzymology*; D013379:Substrate Specificity; D013816:Thermodynamics; D013997:Time Factors
D004791:Enzyme Inhibitors; D010455:Peptides; C072568:methionine 4-nitroanilide; D008715:Methionine; D000626:Aminopeptidases; D063208:Methionyl Aminopeptidases