Catalytically competent human and bovine ζ‐thrombin and chimeras generated from unfolded polypeptide chains

Abstract

Human and bovine α‐thrombin cleaved at the B‐chain by chymotrypsin generates catalytically competent ζ‐thrombins, which are comprised of two noncovalently linked fragments: a 36‐ (human) or 49‐ (bovine) residue A‐chain linked by a disulfide to B‐chain residues B1–148 (ζ1‐thrombin) and B‐chain residues B149–259 (ζ2‐thrombin). Human and bovine d‐Phe‐Pro‐Arg‐CH2‐ζ‐ and PhMeSO2‐ζ‐thrombins were prepared by reaction of the active‐site histidine (H‐B43) and serine (S‐B205) with PPACK and PMSF, respectively. Unfolding and dissociation of the noncovalently linked polypeptide chains of either human or bovine d‐Phe‐Pro‐Arg‐CH2‐ζ‐ and PhMeSO2‐ζ‐thrombins in 4.5 M guanidine‐HCl and refolding upon 30‐fold dilution in 50 mM sodium phosphate buffer pH 6.5, 750 mM NaCl, 0.1% PEG resulted in biphasic generation of catalytic activity. The slow phase was eliminated in the presence of the competitive inhibitor benzamidine‐HCl. Unfolding and refolding mixtures of the appropriate inactive precursors generated the active chimeric thrombins bovine ζ1‐thrombin:human ζ2‐thrombin and human ζ1‐thrombin:bovine ζ2‐thrombin. Human ζ1‐thrombin and ζ2‐thrombin were isolated, and, upon recom‐bining, the isolated fragments refolded to generate catalytically competent ζ‐thrombin with an active‐site content, specific activity toward Chromozym‐TH, and a specificity constant (kcat/Km) for FPA release from fibrinogen that were all within 60% of those of native α‐thrombin. Both α‐ and ζ‐thrombin refolded via first‐order processes (k = 0.011–0.014 s−1), which in the case of ζ‐thrombin were independent of whether ζ1‐thrombin and/or ζ2‐thrombin was incubated in refolding buffer prior to mixing. This observation, together with others, is consistent with the view that generation of catalytically competent enzyme proceeds via a kinetic pathway wherein ζ1‐thrombin and ζ2‐thrombin independently partially refold and form a noncovalent complex that undergoes a rate‐determining rearrangement to active thrombin. Copyright © 1992 The Protein Society