The effects of reversible freezing inactivation and inhibitor binding on redox properties of L-amino-acid oxidase.
We measured the redox potentials of frozen inactivated L-amino-acid oxidase (L-amino-acid:oxygen oxidoreductase (deaminating), EC 1.4.3.2) and inhibitor-bound (anthranilic acid) enzyme, and compared these redox properties to those of active L-amino-acid oxidase and benzoate-bound D-amino-acid oxidase (EC 1.4.3.3), respectively. The redox properties of the inactive enzyme are similar to the properties of free flavin; the potential is within 0.015 V of free flavin and no radical stabilization is seen. This corresponds to the loss of most interactions between apoprotein and flavin. In contrast, the anthranilic acid lowers the amount of radical stabilized from 85% to 35%. The potentials are still 0.150 V positive of free flavin, indicating that in the presence of inhibitor, many flavin-protein interactions remain intact. The difference between this behavior and that of D-amino-acid oxidase bound to benzoate, where the amount of radical declined from 95% to 5%, is explained on the basis of the relative tightness of binding of apoprotein to FAD. D-Amino-acid oxidase apoprotein has a relatively low Ka (10(6)) for FAD, and benzoate has a relatively high Ka (10(5)) for the enzyme. Therefore, the binding of benzoate increases the tightness of FAD binding to apo-D-amino-acid oxidase (10(11)), indicating significant changes in flavin-protein interactions. In contrast, apo-L-amino-acid oxidase binds flavin tightly (the Ka is greater than 10(7)) and the enzyme binds to anthranilate much less tightly, with a Ka of 10(3). The L-amino-acid oxidase apoprotein binding to FAD is tight initially, and the binding of anthranilate changes it only slightly.(ABSTRACT TRUNCATED AT 250 WORDS)
D000594:Amino Acid Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D002942:Circular Dichroism; D005615:Freezing; D050830:L-Amino Acid Oxidase; D010084:Oxidation-Reduction; D011199:Potentiometry; D012910:Snake Venoms / Q000032:analysis; D013057:Spectrum Analysis; D062367:ortho-Aminobenzoates / Q000378:metabolism / Q000494:pharmacology
D012910:Snake Venoms; D062367:ortho-Aminobenzoates; C031385:anthranilic acid; D000594:Amino Acid Oxidoreductases; D050830:L-Amino Acid Oxidase