Activation of a flavoprotein by proteolysis.
Chymotryptic digestion of brain pyridoxine-5-P oxidase brings about a 4-fold enhancement of the catalytic power (Vmax/KM) using pyridoxine-5-P as substrate in the assay mixtures. The chymotrypsin-treated enzyme is less susceptible to inhibition by pyridoxal-5-P than the native enzyme. Fragments arising from limited proteolysis were separated by affinity chromatography using P-pyridoxal-Sepharose as supporting matrix. Catalytically active fractions, eluted by pyridoxine-5-P (5mM), displayed three bands when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular masses of the three protein bands are considerably lower than 28 kDa, the molecular mass of monomeric pyridoxine-5-P oxidase. Spectroscopic studies, absorption, fluorescence, and circular dichroism revealed that the microenvironment surrounding the cofactor flavin mononucleotide is not perturbed by limited proteolysis.
Department of Biochemistry, University of Tennessee, Knoxville 37996-0840.
Y T
YT
Kim
J E
JE
Churchich
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002918:Chymotrypsin / Q000378:metabolism; D004789:Enzyme Activation; D005420:Flavoproteins / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000378:metabolism*; D011734:Pyridoxaminephosphate Oxidase / Q000302:isolation & purification / Q000378:metabolism*; D012756:Sheep; D013053:Spectrophotometry
D005420:Flavoproteins; D011734:Pyridoxaminephosphate Oxidase; D000587:Oxidoreductases Acting on CH-NH Group Donors; D002918:Chymotrypsin