Isolation and characterization of carboxypeptidase III from germinating triticale grains.
Carboxypeptidase III from germinating triticale grains was purified 434.2-fold with a six-step procedure including: homogenization, ammonium sulfate precipitation, cation-exchange chromatography on CM-cellulose, gel filtration chromatography on Sephadex G-150, cation-exchange chromatography on SP8HR column (HPLC), and affinity chromatography on CABSSepharose 4B. Triticale carboxypeptidase III is a monomer with a molecular weight of 45 kDa, which optimally hydrolyzes peptides at temperature 30-50 degrees C and pH 4.6. N-CBZ-Ala-Phe, N-CBZ-Ala-Leu, and N-CBZ-Ala-Met are hydrolyzed at the highest rates. Amino acids with aromatic or large aliphatic side chains are preferred in position P1', whereas the presence of these types of groups in position P1 of the substrate results in a lower rate of hydrolysis. Peptides containing glutamic acid in positions P1 are poor substrates for the enzyme. This phenomenon suggests the hydrophobic substrate- binding sites S1 and S1'. The active site contains serine since diisopropylfluorophosphate and phenylmethanesulfonyl fluoride reduce the activity by 89.9% and 81.5%, respectively. Moreover, the activity of triticale carboxypeptidase III is reduced by mercury ions and organomercurial compounds, which suggests the presence of a sulfhydryl group adjacent to the active site of the enzyme. Identification of purified enzyme by mass spectrometry method demonstrated that the enzyme is a homolog of barley carboxypeptidase III.
Drzymała
Adam
A
Department of Biochemistry, Warsaw University of Life Sciences-SGGW, Warsaw, Poland. adam_drzymala@sggw.pl
Bielawski
Wiesław
W
D000595:Amino Acid Sequence; D002268:Carboxypeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D020134:Catalytic Domain; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D018525:Germination; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature; D014908:Triticum / Q000201:enzymology* / Q000254:growth & development
D011480:Protease Inhibitors; D002268:Carboxypeptidases