PDBsum entry 1gi2

Hydrolase

PDB id: 1gi2

Contents

Protein chain

223 a.a. *

Ligands

SO4

122

Metals

_CA

Waters ×813

* Residue conservation analysis

PDB id:

1gi2

Name:

Hydrolase

Title:

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

Structure:

Beta-trypsin. Chain: a. Ec: 3.4.21.4

Source:

Bos taurus. Cattle. Organism_taxid: 9913. Organ: pancreas

Resolution:

1.38Å R-factor: 0.184 R-free: 0.205

Authors:

B.A.Katz,K.Elrod,C.Luong,M.Rice,R.L.Mackman,P.A.Sprengeler,J.Spencer, J.Hatayte,J.Janc,J.Link,J.Litvak,R.Rai,K.Rice,S.Sideris,E.Verner, W.Young

Key ref:

B.A.Katz et al. (2001). A novel serine protease inhibition motif involving a multi-centered short hydrogen bonding network at the active site. J Mol Biol, 307, 1451-1486. PubMed id: 11292354 DOI: 10.1006/jmbi.2001.4516

Date:

22-Jan-01 Release date: 22-Jan-02

Protein chain

P00760  (TRY1_BOVIN) -  Serine protease 1 from Bos taurus

Seq: 246 a.a.

Struc: 223 a.a.

Enzyme reactions

• Enzyme class: E.C.3.4.21.4 - trypsin.
• Reaction: Preferential cleavage: Arg-|-Xaa, Lys-|-Xaa.

DOI no: 10.1006/jmbi.2001.4516

J Mol Biol 307:1451-1486 (2001)

PubMed id: 11292354

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

B.A.Katz, K.Elrod, C.Luong, M.J.Rice, R.L.Mackman, P.A.Sprengeler, J.Spencer, J.Hataye, J.Janc, J.Link, J.Litvak, R.Rai, K.Rice, S.Sideris, E.Verner, W.Young.

ABSTRACT

We describe a new serine protease inhibition motif in which binding is mediated by a cluster of very short hydrogen bonds (<2.3 A) at the active site. This protease-inhibitor binding paradigm is observed at high resolution in a large set of crystal structures of trypsin, thrombin, and urokinase-type plasminogen activator (uPA) bound with a series of small molecule inhibitors (2-(2-phenol)indoles and 2-(2-phenol)benzimidazoles). In each complex there are eight enzyme-inhibitor or enzyme-water-inhibitor hydrogen bonds at the active site, three of which are very short. These short hydrogen bonds connect a triangle of oxygen atoms comprising O(gamma)(Ser195), a water molecule co-bound in the oxyanion hole (H(2)O(oxy)), and the phenolate oxygen atom of the inhibitor (O6'). Two of the other hydrogen bonds between the inhibitor and active site of the trypsin and uPA complexes become short in the thrombin counterparts, extending the three-centered short hydrogen-bonding array into a tetrahedral array of atoms (three oxygen and one nitrogen) involved in short hydrogen bonds. In the uPA complexes, the extensive hydrogen-bonding interactions at the active site prevent the inhibitor S1 amidine from 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 range 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 suggest 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)benzimidazole 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 inhibitor 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, approximately 6.8. By comparing inhibition constants (K(i) values), inhibitor solubilities, inhibitor conformational energies and corresponding structures of short and normal hydrogen bond-mediated complexes, we have estimated the contribution of the short hydrogen bond networks to inhibitor affinity ( approximately 1.7 kcal/mol). The structures and K(i) values associated with the short hydrogen-bonding motif are compared with those corresponding to an alternate, Zn(2+)-mediated inhibition motif at the active site. Structural differences among apo-enzymes, enzyme-inhibitor and enzyme-inhibitor-Zn(2+) complexes are discussed in the context of affinity determinants, selectivity development, and structure-based inhibitor design.

Selected figure(s)

Figure 4.
Figure 4. Chemical structures of serine protease inhibitor analogs of APC-1144 that (A) make or (B) do not make short hydrogen bonds at the active site.

Figure 6.
Figure 6. (a) Structure and (2|F[o]| - |F[c]|), a[c] map contoured at 1.0 and 2.4s for P2[1]2[1]2[1] (form b) trypsin-APC-1144-Zn2+, pH 8.10, 1.37 Å resolution. Coordinate bonds to the Zn2+ are cyan, and hydrogen bonds are white. (b) Superposition of trypsin-APC-1144-Zn2+ (standard atom coloring scheme) onto trypsin-APC-1144 (carbon, cyan; oxygen, orange; and nitrogen, pink) shows a considerable change in the position and orientation of the phenol ring of the inhibitor. Coordinate and hydrogen bonds in the former are yellow, hydrogen bonds in the latter are cyan.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2001, 307, 1451-1486) copyright 2001.

Figures were selected by an automated process.