The role of SH and S-S groups in Bacillus cereus beta-amylase.
The properties of sulfhydryl (SH) and disulfide (S-S) groups in Bacillus cereus BQ10-S1 Spo III beta-amylase have been investigated to clarify their roles in the enzyme action. Two out of three cysteine residues in B. cereus beta-amylase were found to form an S-S bond, which was found to be located between Cys91 and Cys99 by the analysis of an S-S containing peptide. The replacement of the soybean beta-amylase model around L3 loop 1 revealed that the S-S bond is located at the root of this flexible loop that moves between open and closed forms during catalysis. The analysis of fluorescence labeled peptides revealed that the remaining free SH group was Cys331. Modification of Cys331 with N-ethylmaleimide or p-chloromercuribenzoic acid (PCMB) caused inactivation of the enzyme. The rate constants for the reactions were consistent with those of Cys343 in soybean enzyme. The binding affinity of the PCMB-modified enzyme to maltose was also decreased. These results indicate that the modification of Cys331, which exists as a free SH group in B. cereus beta-amylase caused inactivation by a similar mechanism to that in the case of Cys343 in soybean beta-amylase as assumed from the sequence homology. This cysteine residue has a common role in beta-amylases irrespective their origin.
Nomura
K
K
Research Institute for Food Science, Kyoto University.
Yoneda
I
I
Nanmori
T
T
Shinke
R
R
Morita
Y
Y
Mikami
B
B
D001409:Bacillus cereus / Q000201:enzymology*; D002942:Circular Dichroism; D003545:Cysteine; D004220:Disulfides; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D017433:Protein Structure, Secondary; D013025:Glycine max / Q000201:enzymology; D013438:Sulfhydryl Compounds; D001614:beta-Amylase / Q000737:chemistry* / Q000378:metabolism*
D004220:Disulfides; D010446:Peptide Fragments; D013438:Sulfhydryl Compounds; D001614:beta-Amylase; D003545:Cysteine