Thermodynamic Criterion for the Conformation of P1 Residues of Substrates and of Inhibitors in Complexes with Serine Proteinases

• Published in: Biochemistry (Easton) 1999-06, Vol.38 (22), p.7142-7150
• Main Authors: Qasim, M. A, Lu, Stephen M, Ding, Jinhui, Bateman, Katherine S, James, Michael N. G, Anderson, Stephen, Song, Jikui, Markley, John L, Ganz, Philip J, Saunders, Charles W, Laskowski, Michael
• Format: Article
• Language: English
• Subjects: Alanine - chemistry; Alanine - metabolism; Amino Acids - chemistry; Amino Acids - metabolism; Animals; Aprotinin - chemistry; Aprotinin - metabolism; Binding Sites; Cattle; Chymotrypsin - chemistry; Chymotrypsin - metabolism; Lysine - chemistry; Lysine - metabolism; Models, Molecular; Nuclear Magnetic Resonance, Biomolecular; Protein Conformation; Proteins; Serine Endopeptidases - chemistry; Serine Endopeptidases - metabolism; Serine Proteinase Inhibitors - chemistry; Serine Proteinase Inhibitors - metabolism; Serpins - chemistry; Serpins - metabolism; Substrate Specificity; Thermodynamics; Trypsin Inhibitor, Kazal Pancreatic - chemistry; Trypsin Inhibitor, Kazal Pancreatic - metabolism; Turkeys
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi990265u

Eglin c, turkey ovomucoid third domain, and bovine pancreatic trypsin inhibitor (Kunitz) are all standard mechanism, canonical protein inhibitors of serine proteinases. Each of the three belongs to a different inhibitor family. Therefore, all three have the same canonical conformation in their combining loops but differ in their scaffoldings. Eglin c (Leu45 at P1) binds to chymotrypsin much better than its Ala45 variant (the difference in standard free energy changes on binding is −5.00 kcal/mol). Similarly, turkey ovomucoid third domain (Leu18 at P1) binds to chymotrypsin much better than its Ala18 variant (the difference in standard free energy changes on binding is −4.70 kcal/mol). As these two differences are within the ±400 cal/mol bandwidth (expected from the experimental error), one can conclude that the system is additive. On the basis that isoenergetic is isostructural, we expect that within both the P1 Ala pair and the P1 Leu pair, the conformation of the inhibitor's P1 side chain and of the enzyme's specificity pocket will be identical. This is confirmed, within the experimental error, by the available X-ray structures of complexes of bovine chymotrypsin Aα with eglin c (lacb) and with turkey ovomucoid third domain (1cho). A comparison can also be made between the structures of P1 (Lys+)15 of bovine pancreatic trypsin inhibitor (Kunitz) (1mtn and 1cbw) and of the P1 (Lys+)18 variant of turkey ovomucoid third domain (1hja), both interacting with chymotrypsin. In this case, the conformation of the side chains is strikingly different. Bovine pancreatic trypsin inhibitor with (Lys+)15 at P1 binds to chymotrypsin more strongly than its Ala15 variant (the difference in standard free energy changes on binding is −1.90 kcal/mol). In contrast, turkey ovomucoid third domain variant with (Lys+)18 at P1 binds to chymotrypsin less strongly than its Ala18 variant (the difference in standard free energies of association is 0.95 kcal/mol). In this case, P1 Lys+ is neither isostructural nor isoenergetic. Thus, a thermodynamic criterion for whether the conformation of a P1 side chain in the complex matches that of an already determined one is at hand. Such a criterion may be useful in reducing the number of required X-ray crystallographic structure determinations. More importantly, the criterion can be applied to situations where direct determination of the structure is extremely difficult. Here, we apply it to determine the conformation of the Lys+ side chain in