Differential scanning calorimetric study of the thermal unfolding of Taka-amylase A from Aspergillus oryzae.


Abstract

The thermally induced unfolding of Taka-amylase A, isolated from Aspergillus oryzae, was studied by differential scanning calorimetry. The experimental curves of excess apparent specific heat vs. temperature showed a single asymmetric peak. Curve resolution indicated that this asymmetry is due to the two-state unfolding of three domains in the molecule, with dissociation of the single tightly bound Ca2+ ion occurring during the unfolding of the last domain. Further indication of the dissociation of the specifically bound Ca2+ during denaturation is afforded by the fact that the temperature of maximal excess specific heat, tm, increases with increasing protein concentration in the absence of added excess Ca2+ and with increasing Ca2+ concentration in the presence of added Ca2+. Experiments in a variety of buffers with different enthalpies of ionization indicated that 11.8 +/- 1.5 protons are lost from the protein during unfolding at pH 7.0. In apparent contradiction of this result, the value of tm was found to be essentially independent of pH in the range pH 7-8. No explanation of this anomaly is available. The enthalpy of unfolding at pH 7 and 62 degrees C in the absence of added Ca2+, corrected for the change in buffer protonation, is 2250 +/- 40 kJ mol-1 (42.5 J g-1), and the permanent change in apparent heat capacity is 36.4 +/- 4.1 kJ K-1 mol-1 (0.687 J g-1). Both of these quantities are unusually large for a globular protein. Study holds ProTherm entries: 3932 Extra Details: asymmetry; two-state unfolding; domains; heat capacity

Submission Details

ID: GEzM88xV

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:23 p.m.

Version: 1

Publication Details
Fukada H;Takahashi K;Sturtevant JM,Biochemistry (1987) Differential scanning calorimetric study of the thermal unfolding of Taka-amylase A from Aspergillus oryzae. PMID:3498514
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
3VX0 2013-09-11 1.5 Crystal Structure of alpha-amylase from Aspergillus oryzae
2GUY 2006-08-15 1.59 Orthorhombic crystal structure (space group P21212) of Aspergillus niger alpha-amylase at 1.6 A resolution
2GVY 2006-08-15 1.8 Monoclinic crystal form of Aspergillus niger alpha-amylase in complex with maltose at 1.8 A resolution
7TAA 1998-11-25 1.98 FAMILY 13 ALPHA AMYLASE IN COMPLEX WITH ACARBOSE
6TAA 1993-10-31 2.1 STRUCTURE AND MOLECULAR MODEL REFINEMENT OF ASPERGILLUS ORYZAE (TAKA) ALPHA-AMYLASE: AN APPLICATION OF THE SIMULATED-ANNEALING METHOD
3VX1 2013-09-11 2.2 Crystal Structure of alpha-Amylase from Aspergillus oryzae
3KWX 2009-12-29 2.4 Chemically modified Taka alpha-amylase
2TAA 1982-10-21 3.0 STRUCTURE AND POSSIBLE CATALYTIC RESIDUES OF TAKA-AMYLASE A

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
94.6 Alpha-amylase A type-1/2 Q02905 AMYA_ASPAW
99.6 Alpha-amylase A type-1/2 P0C1B4 AMYA3_ASPOR
99.8 Alpha-amylase A type-1/2 P30292 AMY_ASPSH
94.6 Alpha-amylase A type-1/2 Q02906 AMYB_ASPAW
100.0 Alpha-amylase A type-1/2 P0C1B3 AMYA1_ASPOR