A novel serine protease inhibition motif involving a multi-centered short hydrogen bonding network at the active site

Citation
Ba. Katz et al., A novel serine protease inhibition motif involving a multi-centered short hydrogen bonding network at the active site, J MOL BIOL, 307(5), 2001, pp. 1451-1486
Citations number
71
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Molecular Biology & Genetics
Journal title
JOURNAL OF MOLECULAR BIOLOGY
ISSN journal
0022-2836 → ACNP
Volume
307
Issue
5
Year of publication
2001
Pages
1451 - 1486
Database
ISI
SICI code
0022-2836(20010413)307:5<1451:ANSPIM>2.0.ZU;2-X
Abstract
describe a new serine protease inhibition motif in which binding is mediate d by a cluster of very short hydrogen bonds (<2.3 <Angstrom>) at the active site. This protease-inhibitor binding paradigm is observed at high resolut ion in a large set of crystal structures of trypsin, thrombin, and urokinas e-type plasminogen activator (uPA) bound with a series of small molecule in hibitors (2-(2-phenol)indoles and 2-(2-phenol)benzimidazoles). In each comp lex there are eight enzyme-inhibitor or enzyme-water-inhibitor hydrogen bon ds at the active site, three of which are very short. These short hydrogen bonds connect a triangle of oxygen atoms comprising O-Ser195(y), a water mo lecule co-bound in the oxyanion hole (H2Ooxy), and the phenolate oxygen ato m of the inhibitor (O6'). Two of the other hydrogen bonds between the inhib itor and active site of the trypsin and uPA complexes become short in the t hrombin counterparts, extending the three-centered short hydrogen-bonding a rray into a tetrahedral array of atoms (three oxygen and one nitrogen) invo lved in short hydrogen bonds. In the uPA complexes, the extensive hydrogen- bonding interactions at the active site prevent the inhibitor S1 amidine fr om forming direct hydrogen bonds with Asp189 because the S1 site is deeper in uPA than in trypsin or thrombin. Ionization equilibria at the active site associated with inhibitor binding are probed through determination and comparison of structures over a wide r ange of pH (3.5 to 11.4) of thrombin complexes and of trypsin complexes in three different crystal forms. The high-pH trypsin-inhibitor structures sug gest that His57 is protonated at pH values as high as 9.5. The pH-dependent inhibition of trypsin, thrombin, uPA and factor Xa by 2-(2-phenol)benzimid azole analogs in which the pK(a) of the phenol group is modulated is shown to be consistent with a binding process involving ionization of both the in hibitor and the enzyme. These data further suggest that the pK(a) of His57 of each protease in the unbound state in solution is about the same, simila r to6.8. By comparing inhibition constants (K-i values), inhibitor solubili ties, inhibitor conformational energies and corresponding structures of sho rt and normal hydrogen bond-mediated complexes, we have estimated the contr ibution of the short hydrogen bond networks to inhibitor affinity (similar to1.7 kcal/mol). The structures and Ki values associated with the short hyd rogen-bonding motif are compared with those corresponding to an alternate, Zn2+-mediated inhibition motif at the active site. Structural differences a mong ape-enzymes, enzyme-inhibitor and enzyme-inhibitor-Zn2+ complexes are discussed in the context of affinity determinants, selectivity development, and structure-based inhibitor design. (C) 2001 Academic Press.