Glu 103 Gln site-directed mutation causes an alteration in physical properties of spinach betaine aldehyde dehydrogenase.
Site-directed mutant of spinach betaine aldehyde dehydrogenase (BADH) was obtained by replacing Glu 103 with Gln 103 and the resulting E 103 Q mutant was expressed in Escherichia coli. The mutant BADH as compared to the wild-type BADH was slightly more sensitive to inhibition by NaCl but less sensitive to inhibition by (NH(4))(2)SO(4). Glycinebetaine (GB) activated the wild-type enzyme but not the mutant enzyme. Stronger inhibition by choline was observed in the mutant enzyme than in the wild-type enzyme whereas the reverse was observed for the inhibition by isovaleraldehyde. The mutant enzyme exhibited a broader temperature optimum than the wild-type enzyme, however, the mutant enzyme appeared to be more heat labile. Both mutant and wild-type enzymes could be protected by NAD(+) against thermal inactivation in a similar manner. However, neither GB nor NaCl could afford protection against thermal inactivation in the mutant enzyme whereas some protection was observed in the wild-type enzyme. Similar pH activity profile was obtained for both mutant and wild-type enzymes. The mutant enzyme was less stable than the wild-type enzyme under the pH range of 5-11. Overall results suggest that the negative charge of Glu 103 at the surface of the spinach BADH plays some roles in the maintenance of the structural integrity of the enzyme.
Department of Biochemistry, Faculty of Science, Research Center for Gene Technology, Chulalongkorn University, Bangkok 10330, Thailand. iaran@sc.chula.ac.th
Aran
A
Incharoensakdi
Takashi
T
Hibino
Teruhiro
T
Takabe
D000445:Aldehyde Oxidoreductases / Q000737:chemistry* / Q000235:genetics; D000447:Aldehydes / Q000737:chemistry; D001622:Betaine / Q000737:chemistry* / Q000378:metabolism; D050728:Betaine-Aldehyde Dehydrogenase; D002794:Choline / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004926:Escherichia coli / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry*; D005973:Glutamine / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D018724:Spinacia oleracea / Q000201:enzymology; D013696:Temperature
D000447:Aldehydes; D005973:Glutamine; D018698:Glutamic Acid; D001622:Betaine; D012965:Sodium Chloride; C032251:isovalerylaldehyde; D000445:Aldehyde Oxidoreductases; D050728:Betaine-Aldehyde Dehydrogenase; D002794:Choline