EFFECTS OF SITE-DIRECTED MUTATIONS IN E. COLI ALKALINE
PHOSPHATASE ON THE ACTIVATION ENERGY FOR HYDROGEN EXCHANGE AT THE
TRYPTOPHAN 109 ENAMINE STUDIED BY HYDROGEN EXCHANGE
C.J. Fischer, J.A. Schauerte, K.C. Wisser, A. Gafni, D.G. Steel
This poster was presented at the 1999 Biophysical Society Meeting in
Baltimore, MD
Abstract
Some proteins exhibit a longer tryptophan phosphorescence lifetime in
deuterated solvents than they do in hydrated solvents. In the case of
E. Coli Alkaline Phosphatase (AP) the data suggests that this is a
direct result of deuteration of the indole NH of the phosphorescing
tryptophan.
We have used this observation to monitor the hydrogen exchange of the
phosphorescing tryptophan, W109, of AP by monitoring changes in this
residue's phosphorescence lifetime. Because this residue lies deeply
buried in the hydrophobic core of the protein and exhibits no pH
dependence for its exchange rate it appears to exchange through an EX1
mechanism and therefore its exchange kinetics are related to the
"breathing" motions of the protein's core. We have investigated the
effects of mutations within the vicinity of W109 and have found
significant alterations in W109's hydrogen exchange kinetics. We have
studied both cavity forming mutations (e.g. Q320G) and those which removed
a hydrogen bond within the core (e.g. Q320L). Some mutations, such as the
removal of a hydrogen bond or the formation of a small cavity, tend to
have a minor effect upon the exchange rates and the activation energy of
the exchange whereas other mutations, which affect the packing geometry of
the hydrophobic core, have significant effects. Changes in the exchange
rate reflect significant changes in conformation and can be interpreted at
the structural level.
Supported by NIA grant AGO9761
Introduction