NMR and ESR studies on human pregnancy zone protein. Comparison with human alpha 2-macroglobulin.
NMR and ESR spectroscopies have been used to examine the plasma protease inhibitor pregnancy zone protein (PZP) and its complex with chymotrypsin. The 1H NMR spectrum of PZP shows relatively few sharp resonances, which, by analogy with human alpha 2-macroglobulin, probably arise from the proteolytically sensitive bait region. Upon reaction with chymotrypsin to form a 1:1 protease.PZP tetramer complex, there is a large increase in the intensity of sharp resonances due to an increase in mobility of these residues. 35Cl NMR has been used to follow binding of zinc and manganese to apo-PZP. Zinc binding causes a linear broadening of the bulk Cl-, consistent with access of Cl- to PZP-bound zinc. Since zinc in the two highest affinity sites in human alpha 2-macroglobulin causes no broadening of Cl-, it is concluded that these zinc sites are absent from PZP. The mobility of chymotrypsin in the PZP.chymotrypsin complex was examined by covalently attaching a nitroxide spin label at the serine residue in the active site of the enzyme and examining the appearance of the ESR spectrum. The chymotrypsin is rigidly held by the PZP to which it is covalently bound. In an analogous experiment performed previously on alpha 2-macroglobulin, chymotrypsin, bound in the presence of methylamine and therefore largely noncovalently bound, was found to be free to rotate inside the cage formed by the protease inhibitor.
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
P
P
Gettins
L
L
Sottrup-Jensen
D000595:Amino Acid Sequence; D002712:Chlorides / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D006801:Humans; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008345:Manganese / Q000378:metabolism; D008969:Molecular Sequence Data; D011257:Pregnancy Proteins / Q000302:isolation & purification; D011485:Protein Binding; D011487:Protein Conformation; D015032:Zinc / Q000378:metabolism; D000511:alpha-Macroglobulins
D002712:Chlorides; D011257:Pregnancy Proteins; D000511:alpha-Macroglobulins; D008345:Manganese; D015032:Zinc