Nuclear Magnetic Resonance and Kinetic Studies of the Catalytic Mechanism of the Serine Proteases

Citation

Kaiser, Robert James, Jr. (1984) Nuclear Magnetic Resonance and Kinetic Studies of the Catalytic Mechanism of the Serine Proteases. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/xg11-xg51. https://resolver.caltech.edu/CaltechTHESIS:11162018-115223569

Abstract

Kinetic and nuclear magnetic resonance experiments on the catalytic mechanism of the serine proteases have been carried out using alpha lytic protease, a bacterial serine protease. ¹³C NMR measurements indicate that His 57, and not Asp 102, is the residue titrating with pK_a 6.7 in the free enzyme. However, when alpha lytic protease is complexed with transition state analogs (peptide aldehydes and benzeneboronic acid), the pK_a of this residue can shift to lower values. This shift can be at least as large as 1.8 pK units, and suggests that the enzyme may behave differently in the presence of substrate, especially in its transition state or intermediate forms, than it does in its absence.

The reaction of elastase with specific peptide p-nitroanilides is biphasic, indicating the buildup of a tetrahedral intermediate in a pre-steady state reaction, followed by linear turonver. The intermediate accumulates to about 20-25% of the total amount of substrate bound to the enzyme. The ability for the substrate to make favorable contacts along an extended portion of the enzyme binding site is important in the observation of the buildup of this intermediate.

¹³C NMR studies on model compounds for the Asp 102-His 57 dyad in water and in DMSO solution indicate that pK_a reversal of the two ionizable groups can occur under conditions of moderate dielectric and high polrity. Overall, the results indicate that the "charge relay" mechanism may be operative in cases where the substrate can make precise extended contacts with the enzyme.

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