The purification and characterization of a Bacillus stearothermophilus methionine aminopeptidase (MetAP).
Methionine aminopeptidase (MetAP) catalyzes the removal of an amino-terminal methionine from a newly synthesized polypeptide. The enzyme was purified to homogeneity from Bacillus stearothermophilus (KCTC 1752) by a procedure that involves heat precipitation and four sequential chromatographs (including DEAESepharose ion exchange, hydroxylapatite, Ultrogel AcA 54 gel filtration, and Reactive red 120 dye affinity chromatography). The apparent molecular masses of the enzyme were 81,300 Da and 41,000 Da, as determined by gel filtration chromatography and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), respectively. This indicates that the enzyme is comprised of two identical subunits. The MetAP specifically hydrolyzed the N-terminal residue of Met-Ala-Ser that was used as a substrate, and exhibited a strong preference for Met-Ala- Ser over Leu-Gly-Gly, Leu-Ser-Phe, and Leu-Leu-Tyr. The enzyme has an optimal pH at 8.0, an optimal temperature at 80 degrees C, and pI at 4.1. The enzyme was heat-stable, as its activity remained unaltered when incubated at 80 degrees C for 45 min. The Km and Vmax values of the enzyme were 3.0 mM and 1.7 mmol/min/mg, respectively. The B. stearothermophilus MetAP was completely inactivated by EDTA and required Co(2+) ion(s) for activation, suggesting the metal dependence of this enzyme
Chung
Jae Min
JM
Department of Biochemistry and Molecular Biology, Hanyang University, Sa-1-dong 1271, Ansan, Kyunggi-do, 425-791, Korea.
Chung
Il Yup
IY
Lee
Young Seek
YS
D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002413:Cations, Divalent / Q000494:pharmacology; D002614:Chelating Agents / Q000494:pharmacology; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D063208:Methionyl Aminopeptidases; D008970:Molecular Weight; D009842:Oligopeptides / Q000737:chemistry; D011480:Protease Inhibitors / Q000494:pharmacology; D021122:Protein Subunits; D013379:Substrate Specificity; D013696:Temperature
D002413:Cations, Divalent; D002614:Chelating Agents; D009842:Oligopeptides; D011480:Protease Inhibitors; D021122:Protein Subunits; D000626:Aminopeptidases; D063208:Methionyl Aminopeptidases