Jones, Melvin O. (1982) The factors important to catalysis by serine proteases: structure of diisopropylfluorophosphate-inhibited bovine trypsinogen refined at 2.1 Å resolution. Master's thesis, California Institute of Technology. http://resolver.caltech.edu/CaltechTHESIS:03292010-075029250
Crystals of bovine trypsinogen inhibited with diisopropylfluorophosphate (DFP) were grown at pH 7 and x-ray intensity data to 2.1 Å resolution were collected. The reflections were assigned the phases determined by Kossiakoff (1977) for the isomorphous native trypsinogen structure. Using difference fourier techniques, the resulting protein structure was refined to a residual of 18.2%. As expected, a comparison between (DIP)-trypsinogen and the native trypsinogen structures shows all the structural differences to be in the catalytic site region. However, the structure of the catalytic region was expected to be quite similar to that of DFP-inhibited bovine trypsin at neutral pH, but there exist some marked differences. In trypsin, one of the isopropyl groups of the inhibitor is analogous to the leaving group of a specific substrate and is hydrolyzed off the inhibitor. In the resulting monoisopropylphosphoryl (MIP)-trypsin, the inhibitor is oriented with a non-esterified phosphoryl oxygen in the oxyanion stabilization site as expected for a tetrahedral intermediate in proteolysis. His 57 Nε2 points toward the phosphoryl oxygen at the leaving group site, and is in a position suitable for proton donation to the leaving group. By contrast, the inhibitor on DIP-trypsinogen remains intact and the presence of electron density at each of the three possible isopropyl locations indicates that the groups of the inhibitor are less specifically oriented. After independent refinement of each of the three possible orientations, the favored orientation was with isopropyl groups near the oxyanion and leaving group sites and the non-esterified phopsphoryl oxygen hydrogen-bonded through two solvent molecules to Ser 214 O. The imidazole ring of His 57 is displaced from its native position at the catalytic site out into the solvent region where it forms hydrogen-bonds to Tyr 94 0 and through a solvent molecule to Ser 214 O. Ser 195 Oγ has moved closer to the position occupied by the His 57 imidazole in native trypsinogen and MIP-trypsin. Both statistical variation in orientation of the inhibitor and movement of Ser 195 Oγ toward the native imidazole site can be explained by the nonfunctionality of the oxyanion binding site in trypsinogen. The difference between DIP-Tgen and MIP-trypsin thus suggests both non-productive transition state or substrate binding and stereochemical incompatability between the tetrahedral intermediate and the native imidazole site as possible contributors to the relative inactivity of the proenzyme. Conversely, and of more general importance, these results emphasize the importance of exact stereochemical alignment in generating a functional catalytic enzyme.
|Item Type:||Thesis (Master's thesis)|
|Degree Grantor:||California Institute of Technology|
|Division:||Chemistry and Chemical Engineering|
|Thesis Availability:||Restricted to Caltech community only|
|Defense Date:||1 October 1981|
|Default Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Tony Diaz|
|Deposited On:||15 Apr 2010 18:16|
|Last Modified:||26 Dec 2012 03:23|
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