Interactions of methoxyamine with pyridoxal-5'-phosphate-Schiff's base at the active site of sheep liver serine hydroxymethyltransferase.
The mechanism of interaction of methoxyamine with sheep liver serine hydroxymethyltransferase (EC 2.1.2.1) (SHMT) was established by measuring changes in enzyme activity, visible absorption spectra, circular dichroism and fluorescence, and by evaluating the rate constant by stopped-flow spectrophotometry. Methoxyamine can be considered as the smallest substituted aminooxy derivative of hydroxylamine. It was a reversible noncompetitive inhibitor (Ki = 25 microM) of SHMT similar to O-amino-D-serine. Like in the interaction of O-amino-D-serine and aminooxyacetic acid, the first step in the reaction was very fast. This was evident by the rapid disappearance of the enzyme-Schiff base absorbance at 425 nm with a rate constant of 1.3 x 10(3) M-1 sec-1 and CD intensity at 430 nm. Concomitantly, there was an increase in absorbance at 388 nm (intermediate I). The next step in the reaction was the unimolecular conversion (1.1 x 10(-3) sec-1) of this intermediate to the final oxime absorbing at 325 nm. The identity of the oxime was established by its characteristic fluorescence emission at 460 nm when excited at 360 nm and by high performance liquid chromatography. These results highlight the specificity in interactions of aminooxy compounds with sheep liver serine hydroxymethyltransferase and that the carboxyl group of the inhibitors enhances the rate of the initial interaction with the enzyme.
Acharya
J K
JK
Department of Biochemistry, Indian Institute of Science, Bangalore.
Prakash
V
V
Rao
A G
AG
Savithri
H S
HS
Rao
N A
NA
D000818:Animals; D001665:Binding Sites; D012696:Glycine Hydroxymethyltransferase / Q000378:metabolism*; D006898:Hydroxylamines; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D011732:Pyridoxal Phosphate; D012545:Schiff Bases; D012756:Sheep
D006898:Hydroxylamines; D012545:Schiff Bases; D011732:Pyridoxal Phosphate; C005214:methoxyamine; D012696:Glycine Hydroxymethyltransferase