Pressure-induced perturbation on the active site of beta-amylase monitored from the sulfhydryl reaction.
We investigated the pressure effect on the conformation of beta-amylase by monitoring the chemical reaction of the unpaired cysteine. Sweet potato beta-amylase is composed of four identical subunits, each of which contains six cysteine residues. These residues are inert to 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) in the native state due to steric hindrance. With the increase of the pressure from 0.1 to 400 MPa, the reactivity of one cysteine out of six residues was enhanced. We have identified that the reacted cysteine residue was Cys345 by the chemical cleavage at the reacted site. The reaction kinetics of Cys345 were pseudo-first-order, and the apparent rate constant was increased from 0.001 to 0.05 min(-)(1) with the increase of pressure from 100 to 400 MPa. The activation volume of the reaction rate was calculated as -24 +/- 2 mL/mol from the slope of the logarithmic plot of the pressure dependence of the rate constant. Hysteresis was not evident in the change of intrinsic fluorescence during the cycle of compression and decompression between 0.1 and 400 MPa, indicating that the tetramer does not dissociate under high pressure. This indicates that the enhancement of the reactivity of Cys345 was caused by the perturbation of local conformation under high pressure. The reaction of Cys345 was also enhanced by low concentrations of GuHCl, suggesting the significant role of hydration-driven fluctuation in the pressure-induced enhancement of the reactivity.
Tanaka
N
N
Department of Polymer Science and Engineering, Kyoto Institute of Technology, Matsugasaki, Sakyo, Kyoto 606-8585, Japan. tanaka@ipc.kit.ac.jp
Mitani
D
D
Kunugi
S
S
D001665:Binding Sites; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D004228:Dithionitrobenzoic Acid / Q000737:chemistry; D004795:Enzyme Stability; D019791:Guanidine / Q000737:chemistry; D011312:Pressure; D011487:Protein Conformation; D017510:Protein Folding; D019657:Solanaceae / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013439:Sulfhydryl Reagents / Q000737:chemistry*; D013816:Thermodynamics; D013861:Thiocyanates / Q000737:chemistry; D001614:beta-Amylase / Q000737:chemistry* / Q000378:metabolism*
D013439:Sulfhydryl Reagents; D013861:Thiocyanates; D004228:Dithionitrobenzoic Acid; D001614:beta-Amylase; D019791:Guanidine; D003545:Cysteine; C008658:2-nitro-5-thiocyanobenzoic acid