Protein Science, Vol 1, Issue 8 998-1006, Copyright © 1992 by Cold Spring Harbor Laboratory Press

ARTICLE

Catalytically competent human and bovine {complex}-thrombin and chimeras generated from unfolded polypeptide chains

S. D. LEWIS, D. V. BREZNIAK, J. W. FENTON-II and J. A. SHAFER
Biological Chemistry Department, Merck Sharp and Dohme Research Laboratories, West Point, Pennsylvania 19486

Human and bovine {alpha}-thrombin cleaved at the B-chain by chymotrypsin generates catalytically competent {complex}-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 ({complex}1-thrombin) and B-chain residues B149-259 ({complex}2-thrombin). Human and bovine D-Phe-Pro-Arg-CH(2)-{complex}- and PhMeSO(2)-{complex}-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-CH(2)-{complex}- and PhMeSO(2)-{complex}-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 {complex}1-thrombin:human {complex}2-thrombin and human {complex}1-thrombin:bovine {complex}2-thrombin. Human {complex}1-thrombin and {complex}2-thrombin were isolated, and, upon recombining, the isolated fragments refolded to generate catalytically competent {complex}-thrombin with an active-site content, specific activity toward Chromozym-TH, and a specificity constant (k(cat)/K(m)) for FPA release from fibrinogen that were all within 60% of those of native {alpha}-thrombin. Both {alpha}-and {complex}-thrombin refolded via first-order processes (k = 0.011-0.014 s(-1)), which in the case of {complex}-thrombin were independent of whether {complex}1-thrombin and/or {complex}2-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 {complex}1-thrombin and {complex}2-thrombin independently partially refold and form a noncovalent complex that undergoes a rate-determining rearrangement to active thrombin.
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