Volume 3, Number 1, p. 2-10
D. E. Khoshtariya, M. Shushanian, R. Sujashvili, M. Makharadze, E. Tabuashvili and G. Getashvili
Laboratory of Biochemical Kinetics and Thermodynamics, Institute of Molecular Biology and Biological Physics, Georgian Academy of Sciences, 12 Gotua St, 380060 Tbilisi, Georgia
Enzymatic activity of a-chymotrypsin in the urea-induced molten-globule-like
state: a combined kinetic/thermodynamic study
Kinetic and thermodynamic properties of a-chymotrypsin were studied in
the presence of a moderate denaturant, urea. The enzymatic activity with
a specific substrate, N-acetyl-L-tyrosine ethyl ester, has been examined
at urea concentrations in the range 0-7 M. The apparent Michaelis constant,
Km(app) of the hydrolytic process increased gradually, ca tenfold, approaching
the value of 5 x 10-3 M at the urea concentration of 6
M. Meanwhile, the catalytic constant, kcat, remained unchanged within the
same range of concentration. At a higher urea concentration (7 M) violation
of Michaelis-Menten (MM) kinetics was observed, showing up as anomalous (extra
curved) kinetic traces. In parallel, alteration of the native state was investigated
through differential scanning calorimetry, which revealed that both the thermal
denaturation enthalpy and the transition temperature gradually decrease with
the increase of urea concentration. The calorimetric melting peak, characteristic
of a cooperative transformation, broadened and disappeared totally at a urea
concentration of 6-7 M (at which, however, the kinetics still follow the
MM pattern). Such a trend in thermodynamic behaviour is characteristic of
the so-called molten globule state. Hence, our results directly point to
the surprising preservation of the enzyme’s functional activity in a substantially
altered non-native state.
Key words: a-chymotrypsin; enzymatic activity; heat denaturation;
kinetic parameters; Michaelis-Menten kinetics; microcalorimetry; molten-globule-like
state; thermodynamic parameters; urea additives