Substrate specificity and variables affecting efficiency of mammalian flavin adenine dinucleotide synthetase.
Substrate specificity and product inhibition have been evaluated by using purified rat liver FAD synthetase (ATP:FMN adenylyltransferase, EC 2.7.7.2), obtained by an improved purification protocol with optimized flavin affinity chromatography. FMN analogues studied fall into three general classifications: those with substitution on the pyrimidinoid ring and nitrogen replacement, those with substitution on the benzenoid ring, and those with N(10) side chain modifications. Substitutions on the pyrimidinoid ring and replacement of nitrogens have the greatest influence on binding to enzyme and FAD formation. When the hydrogen-bonding capacity of the NH group at position 3 is blocked or removed by substitution, such FMN analogues do not act as substrates or inhibitors of the enzyme. Substitutions on the benzenoid ring by small groups seem to be tolerated, while larger groups inhibit binding. Length of the N(10) side chain is optimal with five carbons and has greatest affinity for the natural ribityl side chain. Affinity matrices show similar binding characteristics in that the N(3)-(carboxymethyl)riboflavin-agarose does not bind enzyme, while agaroses linked to the flavin N(10) side chain provide varying degrees of purification. The C = O group at position 2, the NH group at position 3, and a five-carbon side chain at the N(10) position seem to be most crucial for flavin substrate binding to enzyme. Nucleoside triphosphates other than ATP do not act as substrates or inhibitors when sufficient Mg2+ is present. Products of the reaction, FAD and PPi, act as inhibitors against both ATP and FMN.(ABSTRACT TRUNCATED AT 250 WORDS)
Bowers-Komro
D M
DM
Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322.
Yamada
Y
Y
McCormick
D B
DB
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives; D000818:Animals; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D005486:Flavin Mononucleotide / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000096:biosynthesis; D008099:Liver / Q000201:enzymology*; D008274:Magnesium / Q000494:pharmacology; D015394:Molecular Structure; D009713:Nucleotidyltransferases / Q000378:metabolism*; D051381:Rats; D013379:Substrate Specificity
D005182:Flavin-Adenine Dinucleotide; D005486:Flavin Mononucleotide; D000255:Adenosine Triphosphate; D009713:Nucleotidyltransferases; C020143:FMN adenylyltransferase; D008274:Magnesium