Proteolytic cleavage of the puromycin-sensitive aminopeptidase generates a substrate binding domain.
The puromycin-sensitive aminopeptidase was found to be resistant to proteolysis by trypsin, chymotrypsin, and protease V8 but was cleaved into an N-terminal 60-kDa fragment and a C-terminal 33-kDa fragment by proteinase K. The two proteinase K fragments remain associated and retained enzymatic activity. Attempts to express the 60-kDa N-terminal fragment in Escherichia coli produced inclusion bodies. A hexa-histidine fusion protein of the 60-kDa N-terminal fragment was solubilized from inclusion bodies with urea and refolded by removal of the urea through dialysis. The refolded protein was devoid of aminopeptidase activity as assayed with arginine-beta-naphthylamide. However, the refolded protein bound the substrate dynorphin A(1-9) with a stoichiometry of 0.5 mol/mol and a K(0.5) value of 50 microM. Dynorphin A(1-9) binding was competitively inhibited by the substrate dynorphin B(1-9), but not by des-Tyr(1)-leucine-enkephalin, a poor substrate for the enzyme.
Ma
Zhangliang
Z
Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, KY 40536, USA.
Daquin
Alex
A
Yao
Jia
J
Rodgers
David
D
Thompson
Michael W
MW
Hersh
Louis B
LB
D000626:Aminopeptidases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D006801:Humans; D006868:Hydrolysis; D015336:Molecular Probe Techniques; D018847:Opioid Peptides / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry; D013379:Substrate Specificity
D018847:Opioid Peptides; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D000626:Aminopeptidases; C025579:enkephalin degrading enzyme; D012697:Serine Endopeptidases