Physics And Chemistry Basis Of Biotechnology - De Cuyper - tiera.ru

Cold-adapted enzymes
flexibility of psychrophilic enzymes. Hydrogen-Deuterium (H/D) exchange offers a
good quantitative parameter for measuring the average conformational flexibility of a
macromolecule. For instance, it has been shown that the thermostable Dglyceraldehyde-3-phosphate
dehydrogenase is more rigid than its mesophilic
counterpart at 25°C, thus suggesting that conformational flexibility is similar at
corresponding physiological temperatures [72]. This fact has been confirmed by H-D
exchange studies performed on thermophilic and mesophilic 3-isopropylmalate
dehydrogenases [71]. Indeed, the thermostable enzyme is more rigid at room
temperature than its mesophilic homologue, whereas the enzymes display nearly
identical flexibility under their respective optimal working conditions. The authors
argued that evolutionary adaptation tends to maintain a “corresponding state” regarding
conformational flexibility: conformational fluctuations necessary for catalytic function
are restricted at room temperature in the thermophilic enzyme, suggesting a close
relationship between conformational flexibility and enzyme function [73]. The fact that
more rigid thermostable proteins reach the flexibility of thermolabile proteins at higher
temperatures was already mentioned by Vihinen and colleagues [74]. In addition, T4lysosyme
site-directed mutagenesis revealed that enzyme residues involved in function
are not optimised for stability, supporting the ‘stability-function’ relationship [75].
Nevertheless H/D exchange rate measurements recorded by Fourier transformed
infrared spectroscopy (FTIR) carried out with psychrophilic and mesophilic α-amylases
failed to reveal any significant difference in the rate and magnitude of amide proton
replacement, indicating either that there is no real difference or that the experiment
temperature was not appropriate. Moreover, loops are well known to be the most
mobile parts of enzyme structures. External loops in the psychrophilic α-amylase are
shorter and the lower bulk of exchangeable protons can possibly compensate for an
improved H/D exchange due to flexibility. NMR experiments on small psychrophilic
enzymes should prove to be an attractive alternative in further investigation of the
expected stability-flexibility relationship. So far, probably the only case in which the
higher flexibility of cold enzymes has been demonstrated is in a fluorescence
spectroscopy experiment on a Ca 2+ -Zn 2+ protease isolated from psychrophilic and
mesophilic Pseudomonas sp. It was clearly shown that the quenching effect of
acrylamide was much more important in the case of the psychrophilic enzyme [43].
However, it has yet to be demonstrated that increased flexibility gives rise to higher
specific activity at low and moderate temperatures.
In fact, the relationship between stability, specific activity and flexibility is much
more complex than was expected. Diverse directed-mutagenesis experiments on
psychrophilic enzymes have led to a better understanding of the activity-flexibilitystability
relationship. In such experiments, weak interactions mediated by residues
occurring in mesophilic or thermophilic enzymes were introduced in psychrophilic
enzymes specifically devoid of these interactions. The main goal of these experiments
was to stabilise the psychrophilic mutants and to record the changes of the specific
activity associated with the mutation. Attention was particularly focused on the αamylase
from the psychrophile Pseudoalteromonas haloplanktis. Among the different
mutations restoring a putative mesophilic character, the double-mutation N 150D-
183