Studies on the inactivation of Leuconostoc mesenteroides NRRL B-512F dextransucrase by o-phthalaldehyde: evidence for the presence of an essential lysine residue at the active site.
The kinetics of inactivation of Leuconostoc mesenteroides NRRL B-512F dextransucrase by o-phthalaldehyde showed that the reaction followed pseudo-first order reaction. The loss of enzyme activity was concomitant with an increase in fluorescence at 417 nm indicating that the inhibition involved the reaction of an epsilon-amino and a thiol group of the enzyme leading to the formation of an isoindole derivative. The stoichiometry of inactivation showed that one isoindole derivative was formed per enzyme molecule. The substrates sucrose and glucose provided protection against o-phthalaldehyde inactivation which was also corroborated by fluorescence studies. Dextransucrase was not inactivated by 5,5'-dithiobis(2-nitrobenzoic acid), showing that the cysteine present in close proximity to the lysine is not essential for enzyme activity. Denaturation of dextransucrase by urea or heat treatment prior to o-phthalaldehyde addition resulted in a decrease of fluorescence intensity indicating that the native conformation of the enzyme is essential for isoindole derivative formation. These results established that a lysine residue is present at the active site and is essential for the activity of dextransucrase.
Goyal
A
A
Department of Chemistry, Indian Institute of Technology Kanpur, India.
Katiyar
S S
SS
D001665:Binding Sites; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005947:Glucose / Q000494:pharmacology; D005964:Glucosyltransferases / Q000037:antagonists & inhibitors* / Q000737:chemistry; D006358:Hot Temperature; D007211:Indoles; D007700:Kinetics; D007934:Leuconostoc / Q000201:enzymology*; D008239:Lysine; D011487:Protein Conformation; D013395:Sucrose / Q000494:pharmacology; D009764:o-Phthalaldehyde / Q000494:pharmacology*
D004791:Enzyme Inhibitors; D007211:Indoles; D013395:Sucrose; D009764:o-Phthalaldehyde; D004228:Dithionitrobenzoic Acid; D005964:Glucosyltransferases; C020025:dextransucrase; D005947:Glucose; D008239:Lysine