Kinetics and mechanism of proteinase-binding of pregnancy zone protein (PZP). Appearance of sulfhydryl groups in reactions with proteinases.
Proteinase binding by pregnancy zone protein (PZP), an alpha-macroglobulin involves bait region cleavages, association of dimeric-PZP into tetrameric and reaction of internal gamma-glutamyl-beta-cysteinyl thiol esters of PZP with proteinase side chains. The product is an equimolar enzyme-PZP(tetramer) covalently linked complex with four free sulfhydryl groups. The kinetics of the appearances of sulfhydryl groups during the reaction of PZP with chymotrypsin has been investigated using stopped-flow and conventional mixing techniques over a broad concentration range. Thiol ester cleavages followed double exponential decays corresponding with two steps. The faster one resulted in the appearance of three sulfhydryl groups with an observed rate constant, k(obs) = k1.1 + k1.2 delta E, dependent on the excess concentration of chymotrypsin, delta E, and k1.1 = 0.03 s-1 and k1.2 = 4 x 10(4) M-1 s-1. The last sulfhydryl group appeared in a slower step, with similar concentration dependence and k2.1 approximately 0.003 s-1 and k2.2 approximately 5 x 10(3) M-1s-1. Covalent binding of the enzyme apparently was simultaneous with the faster thiol ester cleavage step. Based on these and previous results a model of the reaction mechanism of the proteinase binding reaction of PZP is proposed. It consists of four major steps: (i) Bait region cleavage of PZP-dimers by the enzyme, (ii) fast association of enzyme-PZP(dimer) species with native PZP or with another enzyme-PZP(dimer) compound resulting in release of one of the associated enzyme molecules (iii) reaction of an average of three thiol esters of the enzyme-PZP(tetramer) intermediate with the associated internal enzyme molecule or with an external one. In this step one enzyme molecule becomes covalently linked to the PZP-(tetramer), three sulfhydryl groups appear and the enzymic activity of the bound enzyme molecule decreases to the level of that of the final complex. (iv) Hydrolysis of the last thiol ester and in the presence of excess enzyme, degradation of enzyme-PZP(tetramer) complexes and formation of fragments some of which are the size of PZP(dimer) with enzyme bound.
Christensen
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U
Department of Chemistry, University of Copenhagen, Denmark.
Sottrup-Jensen
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L
Simonsen
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M
D000595:Amino Acid Sequence; D001665:Binding Sites; D002918:Chymotrypsin / Q000378:metabolism*; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D010450:Endopeptidases / Q000378:metabolism*; D005260:Female; D006801:Humans; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D011247:Pregnancy / Q000097:blood*; D011257:Pregnancy Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000032:analysis
D009842:Oligopeptides; D011257:Pregnancy Proteins; D013438:Sulfhydryl Compounds; D004228:Dithionitrobenzoic Acid; D010450:Endopeptidases; D002918:Chymotrypsin