Chemical modification of cyclomaltodextrin glucanotransferase from Bacillus circulans var. alkalophilus.
Counting of integral numbers of cysteine residues of the reduced and denaturated form of cyclomaltodextrin glucanotransferase (CGTase) from Bacillus circulans var. alkalophilus (ATCC 21783) showed two cysteine residues per enzyme molecule. Titrations of the enzyme with 5,5'-dithiobis-(2-nitrobenzoic acid) led to the same result. No free SH-group was detected in denatured form of CGTase, indicating that the two cysteine residues are linked by one disulfide bridge. Cyclizing activity of the GdmCl-denaturated and reduced enzyme was 13% of that of the native one. Incubation of CGTase with diethylpyrocarbonate (DEP) showed a pseudo-first-order inhibition with second-order rate constant of 3.2 M-1 s-1. Reaction with hydroxylamine and spectroscopic studies implied that inactivation of CGTase by DEP is due to modification of one histidine residue concomitantly with a 50% decrease in the cyclizing activity (t1/2 = 10.8 min). The inhibition was partially reversible. CGTase was protected against inactivation by alpha- and beta-cyclodextrins suggesting that the modified histidine residue is at or near the active site. Conversion of starch with DEP-modified enzyme resulted in a decreased formation of cyclodextrins while the relative amount of reducing sugars increased. Preliminary results on modification of CGTase with other reagents, e.g., Woodward's reagent K, 2,3-butanedione and carbodiimide are included.
Mattsson
P
P
Department of Biochemistry, University of Turku, Finland.
Pohjalainen
T
T
Korpela
T
T
D003545:Cysteine / Q000032:analysis*; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004220:Disulfides; D004926:Escherichia coli / Q000201:enzymology; D005964:Glucosyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D007700:Kinetics; D010084:Oxidation-Reduction; D011489:Protein Denaturation
D004220:Disulfides; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase; D003545:Cysteine; D004047:Diethyl Pyrocarbonate