Inhibition of human α-thrombin by a phosphonate tripeptide proceeds via a metastable pentacoordinated phosphorus intermediate

X-ray crystallographic studies of human α-thrombin with a novel synthetic inhibitor, an acyl (α-aminoalkyl)phosphonate, reveal the existence of a pentacovalent phosphorus intermediate state. Crystal structures of the complex of α-thrombin with the phosphonate compound were determined independently using crystals of different ages. The first structure, solved from a crystal less than seven days old, showed a pentacoordinated phosphorus moiety. The second structure, determined from a crystal that was 12 weeks old, showed a tetracoordinated phosphorus moiety. In the first structure, a water molecule, made nucleophilic by coordination to His57 of α-thrombin, is bonded to the pentacoordinated phosphorus atom. Its position is approximately equivalent to that occupied by the water molecule responsible for hydrolytic deacylation during normal hydrolysis. The pentacoordinated phosphorus adduct collapses to give the expected pseudo tetrahedral complex, where the phosphorus atom is covalently bonded to Ser195 Oγ. The crystallographic data presented here therefore suggest that the covalent bond formed between the inhibitor’s phosphorus atom and Oγ of Ser195 proceeds via an addition-elimination mechanism, which involves the formation of a pentacoordinate intermediate.