Essential histidyl residues at the active site(s) of sucrose-phosphate synthase from Prosopis juliflora.
Chemical modification of sucrose-phosphate synthase (EC 2.4.1.14) from Prosopis juliflora by diethyl pyrocarbonate (DEP) and photo-oxidation in the presence of rose bengal (RB) which modify the histidyl residues of the protein resulted in the inactivation of the enzyme activity. This inactivation was dependent on the concentration of the modifying reagent and the time of incubation and followed pseudo-first order kinetics. For both the reagents, the inactivation was maximum at pH 7.5, which is consistent with the involvement and presence of histidine residues at the active site of the enzyme. Substrates, UDPG and F6P protected the enzyme against the inactivation by the modifying reagents suggesting that the histidine residues may be involved in the binding of these substrates and are essential for the catalytic activity. Specificity of DEP was indicated by an increase in absorbance at 240 nm along with concomitant inactivation of the enzyme and reactivation of the modified enzyme by hydroxylamine. These results strongly suggest the presence of histidine residue(s) at or near the active site of the enzyme.
Sinha
A K
AK
National Botanical Research Institute, Lucknow 226 001, India.
Pathre
U V
UV
Sane
P V
PV
D000595:Amino Acid Sequence; D001665:Binding Sites; D004047:Diethyl Pyrocarbonate / Q000378:metabolism; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005636:Fructosephosphates / Q000494:pharmacology; D005964:Glucosyltransferases / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D019811:Hydroxylamine / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010940:Plant Proteins / Q000737:chemistry*; D010944:Plants / Q000201:enzymology*; D011732:Pyridoxal Phosphate / Q000494:pharmacology; D012395:Rose Bengal / Q000494:pharmacology; D013053:Spectrophotometry; D014532:Uridine Diphosphate Glucose / Q000494:pharmacology
D004791:Enzyme Inhibitors; D005636:Fructosephosphates; D010940:Plant Proteins; D012395:Rose Bengal; D019811:Hydroxylamine; D006639:Histidine; D011732:Pyridoxal Phosphate; C027618:fructose-6-phosphate; D004228:Dithionitrobenzoic Acid; D005964:Glucosyltransferases; C022698:sucrose-phosphate synthase; D004047:Diethyl Pyrocarbonate; D004229:Dithiothreitol; D014532:Uridine Diphosphate Glucose