Effect of Na+ ions on pH-dependent conformational changes in brush border sucrase-isomaltase in mice intestine.
Intestinal brush border sucrase-isomaltase (sucrose D-glucosidase E.C. 3.2.1.48, E.C. 3.2.1.10) exhibits pH-dependent stimulatory or inhibitory effects in response to Na+ ions. However, whether the enzyme undergoes conformational modulations as a function of pH and in the presence of alkali metal ions is not known. In this paper, we investigated the structural and functional relationship of purified murine sucrase in response to pH and Na+ ions using UV-CD fluorescence and spectroscopic studies. Kinetic studies revealed that at pH 5.0, the enzyme activation by Na+ ions was V-type, which changed to K-type at pH 7.2, whereas at alkaline pH (8.5), Na+ ions inhibited the enzyme activity and inhibition was uncompetitive in nature, affecting both the Km and Vmax components. Far UV-CD spectra of protein at pH 7.2 in the absence and presence of Na+ were almost overlapping, suggesting that secondary structure of protein was not affected upon addition of the salt. However, near UV-CD spectra indicated marked alterations in the tertiary structure of protein in presence of 50 mM Na+ ions. Increase in pH from 7.2 to 8.5 resulted in a marked rise in fluorescence intensity and red shift in lambda max due to tryptophan residues in the enzyme molecule. These findings suggested that alterations in enzyme activity as a function of pH and Na+ ions was associated with ionization of key amino acid residues together with structural modifications in the enzyme conformation around neutral or alkaline pH.
Gupta
Shiffalli
S
Department of Biochemistry, Panjab University, Chandigarh 160014, India.
Mahmood
Safrun
S
Khan
Rizwan Hasan
RH
Mahmood
Akhtar
A
D000818:Animals; D002414:Cations, Monovalent; D002942:Circular Dichroism; D006863:Hydrogen-Ion Concentration; D007413:Intestinal Mucosa / Q000201:enzymology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008871:Microvilli / Q000201:enzymology*; D017434:Protein Structure, Tertiary; D012964:Sodium / Q000737:chemistry*; D013393:Sucrase / Q000737:chemistry; D013394:Sucrase-Isomaltase Complex / Q000737:chemistry*
D002414:Cations, Monovalent; D012964:Sodium; D013394:Sucrase-Isomaltase Complex; D013393:Sucrase