Purification, Characterization, and Substrate and Inhibitor Structure−Activity Studies of Rat Liver FAD-AMP Lyase (Cyclizing):  Preference for FAD and Specificity for Splitting Ribonucleoside Diphosphate-X into Ribonucleotide and a Five-Atom Cyclic Phosphodiester of X, either a Monocyclic Compound or a cis-Bicyclic Phosphodiester−Pyranose Fusion

An enzyme with FAD-AMP lyase (cyclizing) activity, splitting FAD to AMP and riboflavin 4‘,5‘-phosphate (cFMN), was recently identified [Fraiz, F., et al. (1998) Biochem. J. 330, 881−888]. Now, it has been purified to apparent homogeneity from a rat liver supernatant, by a procedure that includes aff...

Full description

Saved in:
Bibliographic Details
Published in:Biochemistry (Easton) 2001-11, Vol.40 (45), p.13710-13722
Main Authors: Cabezas, Alicia, Pinto, Rosa María, Fraiz, Francisco, Canales, José, González-Santiago, Santiago, Cameselle, José Carlos
Format: Article
Language:English
Subjects:
Adenosine Diphosphate - chemistry
Adenosine Diphosphate - pharmacology
Adenosine Triphosphate - chemistry
Adenosine Triphosphate - pharmacology
Animals
Cations - metabolism
Electrophoresis, Polyacrylamide Gel
Enzyme Inhibitors - chemistry
Enzyme Inhibitors - pharmacology
Female
Flavin-Adenine Dinucleotide - chemistry
Flavin-Adenine Dinucleotide - metabolism
Galactose - chemistry
Galactose - metabolism
Glucose - chemistry
Glucose - metabolism
Hydrogen-Ion Concentration
Kinetics
Liver - enzymology
Molecular Weight
Phosphorus-Oxygen Lyases
Protein Renaturation
Pyrophosphatases - antagonists & inhibitors
Pyrophosphatases - isolation & purification
Pyrophosphatases - metabolism
Rats
Rats, Wistar
Structure-Activity Relationship
Substrate Specificity
Uridine Diphosphate - chemistry
Uridine Diphosphate - pharmacology
Xylose - chemistry
Xylose - metabolism
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:An enzyme with FAD-AMP lyase (cyclizing) activity, splitting FAD to AMP and riboflavin 4‘,5‘-phosphate (cFMN), was recently identified [Fraiz, F., et al. (1998) Biochem. J. 330, 881−888]. Now, it has been purified to apparent homogeneity from a rat liver supernatant, by a procedure that includes affinity for ADP-agarose (adsorption required the activating cation Mn2+ and desorption required its removal), to a final activity of 2.2 units/mg after a 240-fold purification with a 15% yield. By SDS−PAGE, only one protein band was observed (M r = 59 000). The correspondence between protein and enzyme activity was demonstrated by renaturation after SDS−PAGE, by gradient ultracentrifugation followed by analytical SDS−PAGE, and by native PAGE with visualization of enzyme activity by fluorescence. A native M r of 100 000 (ultracentrifugation) or 140 000 (gel filtration) indicated that FAD-AMP lyase could be a dimer. The enzyme required millimolar concentrations of Mn2+ or Co2+, exhibited different optimum pH values with these cations (pH 8.5 or 7.3, respectively), and was strongly inhibited by ADP or ATP, but not by dADP, dATP, or the reaction products AMP and cFMN. A specificity study was conducted with 35 compounds related to FAD, mostly nucleoside diphosphate-X (NDP-X) derivatives. Besides FAD, the enzyme split 11 of these compounds with the pattern NDP-X → NMP + PX. Structure−activity correlations of substrates, nonsubstrates, and inhibitors, and the comparison of the enzymic reactivities of NDP-X compounds with their susceptibilities to metal-dependent chemical degradation, pinpointed the following specificity pattern. FAD-AMP lyase splits ribonucleoside diphosphate-X compounds in which X is an acyclic or cyclic monosaccharide or derivative bearing an X-OH group that is able to attack internally the proximal phosphorus with the geometry necessary to form a PX product, either a five-atom monocyclic phosphodiester or a cis-bicyclic phosphodiester−pyranose fusion. For instance, NDP-glucose and GDP-α-l-fucose were substrates, but dTDP-glucose, NDP-mannose, and GDP-β-l-fucose were not. Judging from k cat/K m ratios, we found the best substrate to be FAD, followed closely by ADP-glucose (k cat/K m only 2-fold lower, but not a physiological compound in mammals), whereas other substrates exhibited 50−500-fold lower k cat/K m values. However, there was no evidence for specific flavin recognition. Instead, what seems to be recognized is the NDP moiety of NDP-X, with a
ISSN:0006-2960
1520-4995
DOI:10.1021/bi0157159