Enzymes hirudin

A 20 amino acid polypeptide [1], bivalirudin (hirulog) is a synthetic version of hirudin. Its amino-terminal D-Phe-Pro-Arg-Pro domain, which interacts with the active site of thrombin, is linked via four Gly residues to a dodecapeptide analogue of the carboxy-terminal of hirudin. Like hirudin, bivalirudin also forms a 1 1 stoichiometric complex with thrombin. Once bound, however, the Arg-Pro bond at the amino-terminal of bivalirudin is cleaved by thrombin, thereby restoring active site functions of the enzyme complexes of a-thrombin [2]. 

Recently, we exploited the unique spectroscopic properties of AW to probe the disulfide-coupled folding of the hirudin N-terminal domain 1-47 and the binding to its the target enzyme, thrombin [90]. Before chemical synthesis, the resolution of the commercially available enantiomeric mixture of AW was carried out by treating the racemic mixture with acetic anhydride and subsequent enantioselective deacylation with immobilized Asperigillus oryzae acylase-I to yield L-AW (Scheme 9-1). Purified L-AW was then reacted with 9-fluorenylmethoxycarbonyl chloride... 

Figure 9-14. Schematic representation of the interaction of the N-terminal tripeptide of Y3NT with thrombin. The strnctnre of Y3NT was modeled on the structure of the hirudin-thrombin complex [4htc.pdb 31]. Relevant NT-thrombin distances, in the 2.5-3.5-A range, are indicated by dashed lines. Of note, w606, which in the wild-type hirudin-thrombin structure connects Tyr3 of the inhibitor to Tyr60a of the enzyme, is well suited as a hydrogen bond donor to stabilize the NT ring system [91].

Fig. 9. Large interfaces in serine protease—protease inhibitor complexes. Trypsin and thrombin are evolutionarily related serine proteases of approximately the same size. The 58-residue PTI binds at the active site and makes with trypsin a standard size interface. Ornithodorin and hirudin are thrombin inhibitors that make a large interface with the enzyme. Ornithodorin is a duplicate of PTI hirudin has an extended C-terminal tail that is disordered in solution.

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