Abstract
In Escherichia coli, quinolinic acid, a precursor of NAD+, is synthesized from L-aspartate and dihydroxyacetone phosphate. This synthesis requires two enzymes, a FAD-containing "B protein" and an "A protein." The B protein has been purified 500-fold from E. coli cells. The enzyme behaves as an L-aspartate oxidase. In the absence of A protein, it converts L-aspartate to oxaloacetate. To our knowledge, no enzyme with this activity has been described previously. The enzyme displays some unusual properties. In its role as B protein in quinolinic acid synthetase, product formation (quinolinic acid) is linear with protein concentration; however, when it functions as an L-aspartate oxidase, product formation (oxaloacetate) is a parabolic function of protein concentration. The L-aspartate oxidase activity also shows marked substrate activation at substrate concentrations above 1.0 mM. The L-aspartate oxidase and B protein activities of the enzyme are inhibited by NAD+, which is competitive with FAD. The immediate reaction product of the enzyme has the same characteristics (rate of decay to oxaloacetate, and condensation with dihydroxyacetone phosphate to form quinolinate) as the unstable reaction product (iminoaspartate) formed from D-aspartate oxidase. A reaction mechanism for the A protein-catalyzed condensation of dihydroxyacetone phosphate and iminoaspartate to form quinolinate is presented.
Highlights
NAD', is synthesized from L-aspartate and dihydroxy- have found that thismammalian B protein activity is identical acetonephosphate
Later we found that the E. coli B protein could be replaced by a protein found in mammalian liver [3]
After the blue dextran-Sepharose column purificationstep, E. coli L-aspartate oxidase is completely dependent on added FAD for enzyme activity (Tables I1 and 111).The apparent KMfor FAD, calculated from the data shown in Fig. 8, is 2.5 mM
Summary
&fateriak-~~-[4-'~C]Asparticacid (7.2 mCi/mmol) and "L-[U14C]asparticacid" (200 mCi/mmol) were from ICN. L-Aspartate Oxidase Is the B Protein of &A Synthetase precipitate was dissolved in %o the volume of the original crude extract of 5 mM KP,, pH 8.0, containing 25%glycerol, and was applied to a Bio-Rad P-6 column (3.5 X 30 cm) which was eluted with the same buffer. For Step 4, any protein which precipitated during dialysis in Step 3 was removed by centrifugation, and the solution was applied to a DEAE-cellulose column (3 X 20 cm) equilibrated with 5 mM KP,, 25% glycerol, pH 8.0. The column was washed with 150 ml of the same buffer and eluted with 150 ml of this buffer containing a linear gradient from 0-0.4 M KCl. Assays for amino acid oxidase activities were carried out using the three different assay methods described under “Experimental Procedures.”.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
