当前位置： SCI文献检索 > BIOCHEMISTRY期刊下所有文献 > 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 phospho

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 phospho

Abstract：

: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 (Mr = 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 Mr 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-alpha-L-fucose were substrates, but dTDP-glucose, NDP-mannose, and GDP-beta-L-fucose were not. Judging from kcat/Km ratios, we found the best substrate to be FAD, followed closely by ADP-glucose (kcat/Km only 2-fold lower, but not a physiological compound in mammals), whereas other substrates exhibited 50-500-fold lower kcat/Km 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 strong preference for ADP-X. Splitting would then depend on the presence of an adequate X-OH group. The possibility that, besides FAD, there could be in mammals other ADP-X substrates of FAD-AMP lyase is discussed, with emphasis placed on some ADP-ribose derivatives.

journal_name

Biochemistry

journal_title

Biochemistry

authors

Cabezas A,Pinto RM,Fraiz F,Canales J,González-Santiago S,Cameselle JC

doi

10.1021/bi0157159

subject

Has Abstract

pub_date

2001-11-13 00:00:00

pages

13710-22

issue

eissn

0006-2960

issn

1520-4995

pii

bi0157159

journal_volume

pub_type

杂志文章

在线工具

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 phospho