The reaction of enantiomerically pure (2S)-N-acetyl-L-alanyl-L-phenylalanyl alpha-chloroethane with gamma-chymotrypsin was studied as a probe of the mechanism of inactivation of serine proteases by peptidyl chloroalkanes. It was determined crystallographically that the peptidyl chloroethane alkylates His57 with retention of configuration at the chiral center, indicating a double displacement mechanism. We think it likely that a Ser195-epoxy ether adduct is an intermediate on the inactivation pathway, although other possibilities have not been disproven. Kinetic data reported by others [Angliker et al. (1988) Biochem. J. 256, 481-486] indicate that the epoxy ether intermediate is not an irreversibly inactivated form of enzyme [a conclusion confirmed experimentally (Prorok et al. (1994) Biochemistry 33, 9784-9790)] and that both ring closure of the tetrahedral intermediate to form the epoxy ether and ring opening by His57 partially limit the first-order rate constant for inactivation, ki. The peptidyl chloroethyl derivative adopts a very different active site conformation from that assumed by serine proteases inactivated by peptidyl chloromethanes. Positioning the chloroethyl derivative into the conformation adopted by chloromethyl derivatives would cause the extra methyl group to make a bad van der Waals contact with the inactivator P2 carbonyl carbon, thereby preventing the formation of the invariant hydrogen bond between the inactivator P1 amide nitrogen and the carbonyl group of Ser214. We conclude that the unusual conformation displayed by the chloroethyl derivative is caused by steric hindrance between the extra methyl group and the rest of the inactivator chain.

Selectively labeled polypeptides comprising the gamma-carboxyglutamic acid (Gla) domain (GD) and helical stack (HS) regions of human protein C (PC), and consisting of amino acid residues 1-47, have been chemically synthesized and their Ca2+ binding properties assessed by [13C]-NMR methods. A total of nine such polypeptides have been studied, each containing one of the Gla residues fully enriched with [13C] at its two gamma-carboxylate carbon atoms. Additions of Ca2+ resulted in readily measurable [13C] chemical shifts, titrations of which were used to obtain apparent dissociation constants for each Gla residue in the presence of all other such residues. The Ca2+ titration data obtained on each of the nine polypeptides showed that Gla residues 6, 16, 25, and 26 were involved in the higher affinity Ca2+ binding sites, whereas the remaining Gla residues, viz., 7, 14, 19, 20, and 29, coordinated Ca2+ more weakly. The results are consistent with conclusions drawn from functional studies obtained with site-directed mutations of individual Gla residues and with the structural model of the GD/HS of human PC. In these cases, Gla residues 6, 16, and 26 served as coordination loci for internally located Ca2+ ions, and GD-related Ca(2+)- and PL-dependent properties of PC and activated PC were dependent on the integrity of these Gla residues.