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The structure of the mutant of bacteriophage T4 lysozyme in which Gly-156 is
replaced by aspartic acid is described. The lysozyme was isolated by screening
for temperature-sensitive mutants and has a melting temperature at pH 6.5 that
is 6.1 degrees C lower than wild type. The mutant structure is destabilized, in
part, because Gly-156 has conformational angles (phi, psi) that are not optimal
for a residue with a beta-carbon. High resolution crystallographic refinement of
the mutant structure (R = 17.7% at 1.7 A resolution) shows that the Gly----Asp
substitution does not significantly alter the configurational angles (phi, psi)
but forces the backbone to move, as a whole, approximately 0.6 A away from its
position in wild-type lysozyme. This induced strain weakens a hydrogen bond
network that exists in the wild-type structure and also contributes to the
reduced stability of the mutant lysozyme. The introduction of an acidic side
chain reduces the overall charge on the molecule and thereby tends to increase
the stability of the mutant structure relative to wild type. However, at neutral
pH this generalized electrostatic stabilization is offset by specific
electrostatic repulsion between Asp-156 and Asp-92. The activity of the mutant
lysozyme is approximately 50% that of wild-type lysozyme. This reduction in
activity might be due to introduction of a negative charge and/or perturbation
of the surface of the molecule in the region that is assumed to interact with
peptidoglycan substrates.
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