Purification and biochemical characterization of methionine aminopeptidase (MetAP) from Mycobacterium smegmatis mc2155.
The methionine aminopeptidase (MetAP) catalyzes the removal of amino terminal methionine from newly synthesized polypeptide. MetAP from Mycobacterium smegmatis mc(2) 155 was purified from the culture lysate in four sequential steps to obtain a final purification fold of 22. The purified enzyme exhibited a molecular weight of approximately 37 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Activity staining was performed to detect the methionine aminopeptidase activity on native polyacrylamide gel. The enzyme was characterized biochemically, using L-methionine p-nitroanilide as substrate. The enzyme was found to have a temperature and pH optimum of 50 degrees C and 8.5, respectively, and was found to be stable at 50 degrees C with half-life more than 8 h. The enzyme activity was enhanced by Mg(2+) and Co(2+) and was inhibited by Fe(2+) and Cu(2+). The enzyme activity inhibited by EDTA is restored in presence of Mg(2+) suggesting the possible role of Mg(2+) as metal cofactor of the enzyme in vitro.
Narayanan
Sai Shyam
SS
Biotechnology Division, National Institute for Interdisciplinary Science and Technology, Trivandrum, Kerala, India.
Ramanujan
Ajeena
A
Krishna
Shyam
S
Nampoothiri
Kesavan Madhavan
KM
D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002122:Calcium Chloride / Q000494:pharmacology; D003035:Cobalt / Q000494:pharmacology; D003300:Copper / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005296:Ferrous Compounds / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D006877:Hydroxamic Acids / Q000494:pharmacology; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D015636:Magnesium Chloride / Q000494:pharmacology; D063208:Methionyl Aminopeptidases; D008970:Molecular Weight; D020102:Mycobacterium smegmatis / Q000201:enzymology*; D010455:Peptides / Q000494:pharmacology; D013696:Temperature
D005296:Ferrous Compounds; D006877:Hydroxamic Acids; D010455:Peptides; D015636:Magnesium Chloride; D003035:Cobalt; C016977:amastatin; D003300:Copper; D000626:Aminopeptidases; D063208:Methionyl Aminopeptidases; C018021:cobaltous chloride; D007930:Leucine; C012211:ubenimex; D002122:Calcium Chloride; C010531:actinonin; C029892:cupric chloride; C029451:ferrous chloride