SOME PROTEIN PROPERTIES IMPORTANT FOR ENZYME CATALYSIS - New Trends in Enzyme Catalysis and Biomimetic Chemical Reactions - page 160

polar and non polar interactions and vibrations in a relatively low molecular weight,
(up to 25 kDa) contribute differently to the experimental heat capacity, and its
temperature derivative: (Khechinashvili et al., 1995). In this case, the
non-polar interactions contribute essentially more than polar interactions and vibrations.
The dependencies of on were described by the equation:

and that for (our estimation) by:

Taking into account the molecular weight of kDa and suggesting
the same tendency for non-polar contributions as for low molecular proteins, the
following values were obtained for the hyperthermophylic enzyme:
and (Likhtenshtein et al., 2000). At the same time, the
experimental values are found to be and
[115], which is markedly lower than the expected ones if only non-polar interactions
had contributed. This estimation demonstrates the decisive role played by polar
interactions in enzyme stability. The lower values of and also indicate a lower

value of the protein globule energy fluctuation parameter i.e. it highlights

globule rigidity.
Physical labeling studies on hyperthermostable shed additional light on
the issue of protein rigidity and intramolecular dynamics. In order to evaluate the
conformational flexibility of the enzyme and the mechanisms behind thermal and
chemical perturbant activation, the protein was modified by spin and fluorescent labels
before its label mobility was monitored by ESR and fluorescence spectroscopies
(Shames et al., 2000; Likhtenshtein et al., 2000). The peripheral terminal groups
located away from the active site for 27 Å were labelled by thiocyanide derivatives of
fluorescein and stilbene (FITS and SITS correspondingly). Furthermore, the SH moiety
which lay at a distance of 19 from the active site, close to the entrance of the active

site tunnel, was modified covalently by the nitroxide spin labels with maleimido (MAR . )
and iodoacetamido (IAR . ) chemically active groups and non-covalently by the hybrid
dansyl -nitroxide probe (DR . ).
The following peculiarities of the hyperthermostable which are
different from that for proteins from mesophylic micro-organisms, were revealed in the
results of the spin labelling experiments.
(1) The modification of and SH groups occurred at a rate that is essentially
lower than that typical for mesophylic proteins. This agrees with the enzyme structural
model showing a solvent exposure of about 1% and 38% for SH and

Å

groups,

respectively.