Structural studies of alpha-lytic protease at sub-Angstrom resolution reveal insights into the mechanisms of serine protease catalysis and kinetic stability.
Description
- Language(s)
-
English
- Published
-
2005.
- Summary
-
For many decades, [alpha]-lytic protease ([alpha]LP), an extracellular bacterial protease secreted by Lysobacter enzymogenes , has served as a model in both mechanistic and structural studies for chymotrypsin-like serine proteases. In order to address questions regarding the catalytic mechanism of serine proteases, I determined three structures of [alpha]LP at ultra-high resolution: (1) the free enzyme at its active pH ([alpha]LP pH 8 ; 0.83Å resolution), (2) [alpha]LP at pH5 ([alpha]LP pH 5 ; 0.82Å resolution), and (3) [alpha]LP bound to a peptidyl boronic acid inhibitor, McOSuc-Ala-Ala-Pro-boroVal ([alpha]LP+boroVal(gol); 0.90Å resolution). The latter two structures provided analogs to the transition states of the catalytic reaction. The unexpected covalent binding of a glycerol molecule to the tetrahedral boronate in ([alpha]LP+boroVal(gol); provided the most accurate and highly-resolved model of the tetrahedral intermediate for acylation (TI 1) determined to date. Structure-specific radiation damage was apparent in electron density for these structures, and special data collection techniques were used to minimize radiation damage observed at the Ser195-boronic acid adduct. A comparison of all three structures elucidated the structural changes that occur during the first step of the catalytic reaction (ES [arrow right] TI 1 ). We propose that upon deprotonation of His57, Ser195 undergoes a conformational change that is responsible for preventing the TI 1 [arrow right] ES back-reaction. Based on precise atomic positions obtained at sub-Ångstrom resolution, we have concluded that the His57...Asp102 interaction is a standard ionic hydrogen bond in both the free enzyme and the acylation transition state, and not a low-barrier hydrogen bond as had been previously debated. Instead, I propose that transition state stabilization by chymotrypsin-like serine proteases is achieved through a network of optimized hydrogen bonds that position the catalytic triad and stabilize the Ser195-substrate tetrahedral adduct. In particular, I identified a short, ionic hydrogen bond between His57 and the amide of the substrate leaving group that may play the primary role in catalyzing the second step of the acylation reaction. A surprising outcome from these studies was the discovery of a site of conformational strain that appears to be evolutionarily linked to [alpha]LP's kinetic stability. Distortion of Phe228, a conserved residue in the protein core, is estimated to account for ~4 kcal/mol in conformational energy. The rearrangement and subsequent distortion of Phe228 by co-evolved residues surrounding it supports the hypothesis that tight packing in [alpha]LP is a key component of [alpha]LP's folding energy landscape. Subsequent mutational studies support this hypothesis, confirming our discovery of the first known functionally-relevant distortion.
- Note
-
Adviser: Agard, David A.
Source: Dissertation Abstracts International, Volume: 66-06, Section: B, page: 3114.
- Physical Description
-
xiv, 218 p. :
col. ill. ;
28 cm
Viewability