K. Ogasahara et al., ELECTROSTATIC STABILIZATION IN METHIONINE AMINOPEPTIDASE FROM HYPERTHERMOPHILE PYROCOCCUS-FURIOSUS, Biochemistry, 37(17), 1998, pp. 5939-5946
The thermostability of methionine aminopeptidase from a hyperthermophi
le P. furiosus (PfMAP) was extremely high: the denaturation temperatur
e was 106.2 degrees C at pH 10.2. To explore the contribution of elect
rostatic interaction to the superior thermostability of PfMAP, the the
rmostability of PfMAP was examined by differential scanning calorimetr
y (DSC) in various salt concentrations in the acidic region far from t
he isoelectric point of PfMAP. (1) In 20 mM glycine buffer, the DSC cu
rve of PfMAP exhibited a single peak. Transition temperatures (T-m) we
re lowered with decreasing pH from 4 to 3. The heat denaturation of Pf
MAP was not reversible. (2) Denaturation enthalpy (Delta H) measured a
t different pHs linearly correlated with T-m up to 102 degrees C, sugg
esting that the denaturation heat capacity (Delta C-p) for pfMAP is co
nstant up to 100 degrees C. Delta C-p was estimated to be 0.82 J K-1 g
(-1). (3) In the presence of 10-100 mM KCl at pH 3.2., two peaks appea
red on the DSC curves. The first peak shifted to lower temperatures wi
th increasing concentration of KCI and, oppositely, the second one to
higher temperatures. It was found that the first and second peaks orig
inated from the heat denaturation of the native form of PfMAP and the
melting of the non-native associated form having molten globule-like s
tructure, respectively, judged from the CD spectra and ultracentrifuga
tion analyses. This indicates the following: first, the attractive ele
ctrostatic interaction is an important factor in stabilizing the nativ
e form of PfMAP; second, the presence of KCl stimulates the formation
of the molten globule-like state of PfMAP and stabilizes it. (4) In a
comparison of the sequence and crystal structure of PfMAP, which has b
een recently determined (1xgs.pdb), with those of MAP from Escherichia
coli (EcMAP), it was predicted that the extra four short-range ion pa
irs less than 3 Angstrom involved in PfMAP are crucial candidates as d
eterminants for the superior thermostability of PfMAP.